JP2003532390A - Compositions and methods for inducing proteins involved in xenobiotic metabolism - Google Patents

Abstract

  (57) [Summary] The present invention provides improved cells and methods for identifying compounds that alter protein expression, such as xenobiotics, endogenous substances, chemicals and drugs. One embodiment of the present invention is directed to a first nucleic acid molecule comprising a promoter or enhancer capable of functioning as a nucleic acid molecule encoding a protein (such as an enzyme or a transporter) involved in drug metabolism and a reporter gene; A second nucleic acid encoding an intracellular receptor or transcription factor, wherein when the intracellular receptor or transcription factor binds to the compound, the intracellular receptor or transcription factor leads to expression of the reporter gene or a promoter or Enable to operably couple to enhancer. Another aspect of the present invention provides a method for evaluating a compound for the property of inducing the expression of a gene encoding a protein involved in drug metabolism, comprising providing a test compound; contacting the test compound with a cell of the present invention; A method comprising detecting expression of a reporter gene. This method can be a high-throughput method.

Description

Detailed Description of the Invention

This application is filed on Apr. 12, 2000, entitled “Method for In Vitro Screening for Drug Metabolism,” US Provisional Patent Application Serial No. 60 / 196,681, and Oct. 17, 2000. Claims the privilege of priority to US Provisional Patent Application Serial No. 60 / 241,391, entitled "Large Screening for p450 Induction," which is incorporated by reference herein in its entirety. .

This invention was made with government support awarded by the National Institutes of Health, Grant No. GM-58287, and it may be said that the US Government has certain rights in the invention.

TECHNICAL FIELD The present invention relates to the field of identifying compounds that alter protein expression.

Background Rejection of therapeutic agents is a common cause of morbidity and mortality, especially in industrialized countries where the use of such therapeutic agents is relatively prevalent. Side effects from drugs were estimated to be the fourth to sixth leading cause of death in US hospitals (Moore and Kliewer, Toxicology).
153: 1-10 (2000)). Many of these rejection reactions are due to drug-drug interactions, whereby the administration of one drug modifies the properties of a second co-administered drug. The most common drug-drug interactions occur when one drug increases or decreases the potency of another (Moore).
and Kliewer, Toxicology 153: 1-10 (2000
)). This change in the pharmacological response of the drug is generally due to changes in the metabolism of the drug. Therefore, a major factor associated with drug-drug interactions is altered metabolism.

The tissues most involved in drug metabolism are the liver and intestine. Within these tissues, oxidative metabolism of drugs and other xenobiotics occurs by the action of the superfamily of heme-containing monooxygenases, collectively known as cytochrome P450 enzymes (CYP's) ( Never and Gonzale
z, Ann. Rev. Biochem. 56: 945-993 (1987)).
In general, the enzymatic action of CYPs leads to the formation of highly polar products, leading to faster clearance of the products with respect to the drug itself. This process can significantly change the pharmacodynamic characteristics of a drug. Such a response is of particular importance when it affects drugs within a narrow therapeutic range.

The most abundant CYP enzyme in human liver and intestine is CYP3A4
And about 70% of the total enterocyte CYPs (Moore and Kliewer,
Toxicology 153: 1-10 (2000)) and liver P450s.
Account for about 29% to about 60% (Wrightton et al., Drug
Metab. Rev. 32: 339-361 (2000)). The substrate of CYP3A4, the microsomal enzyme, is generally highly lipophilic. Known CYP3A4
Substrate structural differentiation is widespread, including endogenous steroids, contraceptive steroids, immunosuppressants, imidazole antifungals and macrolide antibiotics (Wrighton
et al. , Drug Metab. Rev. 32: 339-361 (20
00)). Due to the abundance of CYP3A4 in the liver and intestine and its broad substrate specificity, CYP3A4 is believed to play a dominant role in the biotransformation of drugs. This P450 enzyme is estimated to be involved in the metabolism of over 50% of all drugs used today (Wrightton et al., Drug Metab.
Rev. 32: 339-361 (2000)).

Prolonged exposure to drugs can lead to increased expression of certain p450s that can increase metabolism and clearance of therapeutic drugs. CYP3A4 activity is enhanced by a range of different drugs, and the induced expression accounts for many drug-drug interactions. Some of the most effective inducers of CYP3A4 expression are widely used drugs, including the glucocorticoid dexamethasone, the antiepileptic drug phenobarbital, the antibiotic rifampicin and the antifungal clotrimazole ( Lehmann et al., J. Clin. Invest.
102: 1016-1023 (1998)). As a result of elevated CYP3A4 levels, the therapies metabolized by this P450 show low efficacy. Therefore, what is important is to identify agents that have the ability to induce drug-metabolizing enzymes, including but not limited to CYP3A4 and other p450 enzymes.

The biochemical properties of P450 regulation can be complex. Several inducers of p450 activity have been identified. CYP3A4 levels were induced by exposure to a number of structurally distinct agents. This diversity makes it difficult to predict new drugs that may affect the expression of that enzyme. Glucocorticoids and other nonsteroidal CYP3A4 inducers transcriptionally drive this P450 expression by a mechanism involving the orphan nuclear receptor, pregnane X receptor (PXR) and potentially other receptors. Adjust. PXR has been identified as a new member of the nuclear hormone receptor superfamily. PXR is CY
It mediates the induction of high doses of glucocorticoid and pregnane steroids of the P3A4 promoter, which form a heterodimer with the nuclear hormone receptor partner RXR, and a highly conserved element in the CYP3A4 promoter, the PXR element (PXRE). By combining. The nucleotide limitation for PXR binding is clearly defined as AGTTCA, which is arranged as a direct repeat (DR) or an inverted repeat (ER) at 3, 4, 5 or 6 nucleotide intervals. (Wrighton et al., Drug Metab. Rev.
． 32: 339-361 (2000)).

Since CYP3A4 and other CYPs may represent species differences, pharmaceutical companies test their drug candidates in vitro in the human system, in order to obtain an assessment of potential drug-drug interactions in humans. Is. Most in vitro studies involve the use of primary cultures of human hepatocytes. The usefulness of hepatocytes has given the drug industry the ability to obtain clinically relevant data on drug-drug interactions in vitro. Compounds identified as potential inducers of human P450 in hepatocytes can be excluded from further development, reducing drug-drug interactions and thus safety and potential for commercial obstruction (Rodrigues, Pharm. Res. 14
: 1504-1510 (1997)).

However, there are also disadvantages of using primary cultures for these tests. One logical problem with hepatocyte preparation is that enzyme activity is not stable for more than about four or five days. Also, these systems are expensive, time consuming, and produce variable responses.
In addition, utility can be sporadic because primary cultures depend on the availability of human organs. The results obtained using such cells also depend on the culture medium and the culture conditions. Finally, the number of compounds that can be tested at any given time is limited. This is particularly problematic because of the large number of candidate drugs produced by combinatorial chemistry and combinatorial biology methods.

Other in vitro systems have been investigated because of the inherent problems associated with the use of human hepatocytes for preclinical drug development and the difficulty of obtaining liver specimens for research purposes. Transcriptional activation has been performed in vitro for many years to investigate the changes in gene expression of P450 enzymes by chemicals (Plant et al.
al. , Analyt. Biochem. 278: 170-174 (2000)
). The most common type is the use of transient transfection of reporter gene constructs into appropriate cell lines. Following this is the administration of the test compound,
Measurement of reporter gene product and comparison with control cells. Biological and experimental variability can occur during each step of this procedure, which can result in poorly reproducible results and possibly incorrect interpretations. Examples of such variations include initial transfection efficiency, activation by endogenous factors on the host cell line, and chemical specific effects such as cytotoxic or proliferative effects. These problems discourage enthusiasm for using these types of systems.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 depicts a series of diagrams for one embodiment of the present invention in which a first nucleic acid molecule and a second nucleic acid molecule are extrachromosomal factors such as plasmids. Is offered as. As shown in FIG. 1A, regulatory factor P2 regulates transcription of genes encoding intracellular receptors or transcription factors. The translation product can then interact with a test compound that binds to intracellular receptors or transcription factors. As shown in FIG. 1B,
The complex of the intracellular receptor or transcription factor with the xenobiotic or test compound can then bind to a promoter or enhancer capable of acting on the nucleic acid molecule encoding the protein involved in drug metabolism. The complex can also enter the nucleus and optionally bind an endogenous promoter or enhancer capable of acting on the nucleic acid molecule encoding the protein involved in drug metabolism, which is such that the complex This is the case when it is present in the cell or is active. After binding this complex to a promoter or enhancer capable of acting on a nucleic acid molecule encoding a protein involved in drug metabolism, the reporter gene is transcribed and translated into a reporter, and the endogenous enzyme involved in drug metabolism is It is optionally expressed when the reporter gene is present or active in such cells (FIG. 1C). A reporter can be a protein that is detectable by physical properties such as fluorescence or luminescence, or a protein that can be detected based on its enzymatic conversion of a substrate into a product, such as a detectable product (FIG. 1D). Such a reporter can be intracellular or extracellular. According to another aspect of the invention, both the first nucleic acid molecule and the second nucleic acid molecule are provided on the same extrachromosomal element, eg a single plasmid or YAC.

FIG. 2 shows the case where the first nucleic acid molecule is the extrachromosomal factor (20), while the second nucleic acid molecule is internal to the chromosome (22) of the cell.

FIG. 3 shows the case where the first nucleic acid molecule is an extrachromosomal factor (30) and the second nucleic acid molecule is an exogenous nucleic acid molecule (32) integrated into the cell's genome. .

FIG. 4 shows that an exogenous nucleic acid molecule (40
) And the second nucleic acid molecule is internal to the chromosome of the cell (42).

FIG. 5 shows that an exogenous nucleic acid molecule (50
) And the second nucleic acid molecule is an exogenous nucleic acid molecule (52) integrated into the cell's genome.

FIG. 6 shows the structure of the HepG2 cell line with a stably integrated CYP3A4 PXRE / luciferase reporter construct. The CYP3A4 PXRE containing the ER6 DNA sequence was constructed in the plasmid pGL3 promoter (Pro
It is equipped with. Transfected cells were cultured in the presence of geneticin and integrated plasmid pIRESneo (Clone).
tech). Geneticin resistant colonies are screened for rifampicin enhanced luminescence.

FIG. 7 shows the response of the HepG2 cell line shown in FIG. New chemical substance (
After binding to a ligand such as NCE), the endogenous HepG2 PXR is activated and forms a heterodimer with the endogenous HepG2 RXR. PXR /
The RXR complex binds to the PXRE sequence cloned into the pGL3-promoter (Promega) and stably integrated within the HepG2 genome. Binding of the PXR / RXR complex activates transcription of the integrated pGL3 promoter plasmid from the SV40 promoter. The luciferase gene is transcribed and translated to cause NCE dose-dependent luminescence.

FIG. 8 shows Northern blot analysis of RNA, which was
Stable transformation with pIRES containing only R (colonies D6, D4, B6
B5, A5, A2, W6, W5, W4, W2 and W1), or from individual colonies stably transformed with the combination of the pGL3 promoters containing the CYP3A4 enhancer element described in the example (Colony 6G). Being separated. Each well contained 10 micrograms of total RNA and was developed with a cDNA probe specific for hPXR.

FIG. 9 shows the effect of rifampicin and DMSO treatment on cells stably transformed with the pGL3-promoter / 3A4 enhancer. 96-well plates were used to determine the length of exposure to cause high levels of luciferase induction. Cells were treated between zero and 78 hours before luciferase activity was measured. Results are expressed as fold increase over DMSO control cells and are the result of four experiments.

[0021] Figure 10 shows the effect of rifampicin treatment on cells containing the pGL3 / 3A4 enhancer plus phPXR. Cells were cultured in 96-well plate format and plated in 10 micromolar rifampicin or DMSO.
It was exposed for 2 hours. Results are expressed as relative luminescence and are the result of four experiments.

FIG. 11 shows hPXR plus pGL3 / 3A4 enhancer or pG
Shown are the results of adding various amounts of cells containing only the L3 / 3A4 enhancer to 96-well plates and treating with 10 micromolar rifampicin or DMSO for 48 hours. Results are expressed as fold increase over control DMSO treated cells and are the result of four experiments.

FIG. 12 shows the effect of serum, DMSO and rifampicin on luciferase activity in HepG2 cells stably transformed with pGL3 vector and pIRES vector with hPXR. Cells were treated for various times ranging from zero to 78 hours with or without 0.1% serum in rifampicin, DMSO, or medium. Additional controls without either rifampicin or DMSO were also included. Results are expressed as relative luminescence and are the result of four experiments.

FIG. 13 shows various CYP3A against CYP3A4 expression in human hepatocytes.
4 shows the effect of the inducer. Human hepatocytes were exposed to 10 micromolar dexamethasone, no dexamethasone, or the amount of dexamethasone normally present in hepatocyte culture medium (approximately 10 ⁻⁷ M). Other inducers include 1
Mmol phenobarbital, 10 μmol rifampicin, clotrimazole, or RU486. Total RNA (10 micrograms) was subjected to Northern blot analysis and as described in the examples, CYP3A4.
Was developed with a cDNA probe specific for.

FIG. 14 shows the effect of various CYP3A4 inducers and non-inducers on HepG2 cells stably transformed with hPXR in pIRES vector and 3A4 enhancer in luciferase vector (colony 1F). Cells are exposed to each inducer in a 96-well plate format for 72 hours before measuring luciferase activity. Cells are 1 micromolar dexamethasone, 100 micromolar omeprazole, 10 micromolar clotrimazole, 10 micromolar RU486, 10 micromolar rifampicin, 100 micromolar mevastatin, 50 micromolar PC.
N, treated with 100 micromolar phenobarbital, 1 micromolar TCDD. Data are expressed as fold increase in luciferase activity relative to control DMSO-treated cells and are the results of four measurements.

FIG. 15 shows various CYP3s for HepG2 cells stably transformed with the 3A4 enhancer in the luciferase vector (colony 13).
The effect of A4 inducer and non-inducer is shown. Luciferase activity is measured after cells have been exposed to each inducer in a 96-well plate format for 72 hours. Cells are 1 micromolar dexamethasone, 100 micromolar omeprazole, 10 micromolar clotrimazole, 10 micromolar RU486, 10 micromolar rifampicin, 100 micromolar mevastatin, 50 micromolar PCN, 100 micromolar. Phenobarbital of 1 micromolar of TCDD. Data is control DMS
The results are shown in fold increase of luciferase activity with respect to O-treated cells, and are the results of four measurements.

FIG. 16 shows various Cs for HepG2 cells stably transformed with the CYP3A4 enhancer in the luciferase vector (colony 13).
Figure 3 shows the effect of different doses of YP inducer. Luciferase activity is measured after cells have been exposed to each inducer in a 96-well plate format for 72 hours. Cells were treated with three times the dose of each drug. Dosages ranged from 0.1 micromolar to 5 millimolar, depending on the agent. Dexamethasone doses are 0.1 micromolar, 1.0 micromolar and 10 micromolar; omeprazole is 50 micromolar, 100 micromolar and 250 micromolar; clotrimazole is 5 micromolar, 10 micromolar and 5 micromolar.
0 micromolar; phenobarbital 1 mmol, 2 mmol and 5 mmol; TCDD 0.5 nanomolar, 1 nanomolar and 2 nanomolar; RU486
Is 5 micromolar, 10 micromolar and 50 micromolar; rifampicin is 5 micromolar, 10 micromolar and 25 micromolar; and mevastatin is 10 micromolar, 50 micromolar and 100 micromolar. Results are expressed as fold increase in luciferase activity relative to DMSO treated cells and the mean of 6 measurements +/- standard deviation. The minimum dose of each drug was shown to increase from left to right.

FIG. 17 shows the effect of culturing stable cell lines in 24 and 96 well plates. 101L cells were cultured in 24 or 96 well plates and exposed to various doses of the Ah receptor ligand benzanthracene. Luciferase activity was assessed after 18 hours of exposure. Results are expressed as the mean +/- SD of 3 different experiments.

FIG. 18 shows response time curves of various CYP1A1 inducers. The maximum time of inducer exposure was determined by establishing the time course of inducer-mediated luciferase activity in 101L cells and in 96 well plates. The enhanced activity was demonstrated by benzanthracene (100 micromol), omeprazole (10
0 micromolar) and 3-methylcholanthrene (10 micromolar) were observed within 6 hours of administration. Cells were also treated with rifampicin (100 micromolar) as a negative control. Each point represents the mean +/- SD of the results of three tests.

FIG. 19 shows response dose curves for various known CYP1A1 inducers.
The effects of various CYP1A1 inducers are shown in 96-well plate format and 10
It was measured using 1 L cells. Dose response curves were generated for TCDD (0.5 to 2.2 nanomolar (panel A), benzanthracene and omeprazole (1 to 200 micromolar) (panel B). Each point is the result of three studies. The mean of +/− SD is shown.

FIG. 20 shows dose response curves for various flavonoids. Using the 96-well plate format and 101L cells, the dose response curve was GTE (
Insert) was created. Doses ranged from 0.01 milligram / ml to 0.2 milligram / ml with 18 hours of exposure. The dose response curve is EGCG
, Quercetin, curcumin, kaempferol, naringenin, apigenin, and resveratrol were also determined and ranged from 1 to 20 micromolar. The exposure to each agent was 18 hours. Each point represents the mean +/- SD of the results of three tests.

FIG. 21 shows the effect of co-processing of TCDD and each flavonoid. CYP1A1-containing cell lines were treated with 10 micromoles of each flavonoid, or 0.1 mg / ml GTE, and 2 nmoles TCDD.
The cells were exposed to both agents for 18 hours. Results are the average of three tests + /
-Indicates SD.

Overview The in vitro system described herein can detect the induction of drug metabolizing enzymes, including P450s such as CYP3A4. The disclosed method can detect xenobiotic transcriptional activation of appropriate enhancers and reporter genes, wherein the enhancers and reporter genes are stable independently and stably in host cells such as human hepatoma cells. It has been transfected. The system can be used in a microtiter plate format and the results can optionally be obtained with a suitable microtiter plate reader within a few days of applying the drug candidate to the cells. The advantages of this in vitro transcription system are many when compared to isolated human hepatocytes or liver slices and include improved assay consistency and reproducibility. Also, inter-individual or inter-species variability and culture conditions that can affect assay results are addressed using the system of the present invention. The system can be formalized for high throughput assays and can predict double or more induction of a gene encoding a particular drug metabolizing protein in a relatively short time.

According to one preferred embodiment of the invention, the in vitro system is essentially high throughput and capable of assessing CYP3A4 induction. This preferred embodiment of the invention comprises the regulatory region of the CYP3A4 gene designated PXRE and the transcription factor PXR. The PXRE is operably linked to a reporter gene such as luciferase, such as on a plasmid. The plasmid containing the PXRE and the reporter gene is then stably transformed into a hepatoma cell line such as HepG2. Once transformed, PXR can bind to PXRE and activate transcription. This can occur when PXR is stimulated by a suitable ligand such as a drug. A nucleic acid molecule encoding a drug-metabolizing protein other than CYP3A4 or P450's can be used by substituting the nucleic acid molecule. Appropriate regulatory regions other than PXRE can also be used, making the regulatory regions suitable for nucleic acid molecules encoding drug metabolizing enzymes or transporters. In addition, a reporter gene other than luciferase, such as a detectable protein, green fluorescent protein (GFP) or variants thereof, or other enzymes such as beta-galactosidase, beta-lactamase or alkaline phosphatase should be used in this system. You can Alternative cells can be used, but cells derived from tissues involved in drug metabolism are recommended.

It is recognized by the present invention that a cell-based system for assessing compound interactions can be created using a suitable nucleic acid molecule, which nucleic acid molecule encodes a reporter gene. Included are nucleic acid molecules encoding proteins involved in drug metabolism operably linked to the molecule, and one or more enhancers or promoters for nucleic acid molecules encoding intracellular receptors or transcription factors. These nucleic acid molecules can be extrachromosomal or can stably integrate into the genome of the cell. In addition, in certain cases, the nucleic acid molecule may be endogenous to the chromosome of the cell, especially when the nucleic acid molecule encodes an intracellular receptor, transporter or transcription factor.

One aspect of the present invention provides a cell, wherein the cell comprises a first nucleic acid molecule: the first nucleic acid molecule is for a nucleic acid molecule encoding a protein involved in drug metabolism. Contains a functional promoter or enhancer and a reporter gene. Preferably, a promoter or enhancer is operably linked to said reporter gene. The cell also comprises a second nucleic acid encoding an intracellular receptor or transcription factor, such that when the intracellular receptor or transcription factor is bound or activated with the compound, the intracellular receptor or transcription factor is Is operably linked to the promoter or enhancer to direct expression of the reporter gene. The reporter gene is expressed when the cell is contacted with a compound that induces the expression of a protein involved in drug metabolism.

A second aspect of the invention provides a method of evaluating a compound for the property of inducing expression of a gene encoding a protein involved in drug metabolism, the method comprising: Providing; contacting a test compound with cells of the invention; and detecting expression of said reporter gene. Expression of the reporter gene indicates that the compound altered the expression of the gene encoding the protein involved in drug metabolism.

[0038]   Detailed Description of the Invention

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The scientific names generally used in this specification and the experimental procedures in cell culture, chemistry, microbiology, molecular biology, cell science and cell culture described below are well known in the art and widely used. Conventional methods are used in these procedures, for example, those provided in the state of the art and various general references (Sambrook et al.
． , Molecular Cloning: A Laboratory Man
ual, 2nd edition, Cold Spring Harbor P
Less, Cold Spring Harbor, N.M. Y. (1989); A
usubel et al. , Current Protocols in M
olecular Biology, John Wiley and Sons
(1998); Harlowe and Lane, Antibodies, a.
Laboratory Manual, Cold Spring Harbo
r Press (1988)). When a term is used in the singular, the inventor also contemplates the plural of the term. The scientific names used in this document and the experimental procedures described below are well known and widely used in this field. As used throughout this specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

A “nucleic acid molecule” is a polynucleotide. The nucleic acid molecule can be DNA, RNA, or a combination of both. Nucleic acid molecules also include sugars other than ribose and deoxyribose incorporated into the backbone and thus can be other than DNA or RNA. Nucleic acids can include nucleobases, which either occur naturally or do not occur naturally, such as xanthine, which is a derivative of a nucleobase such as 2-aminoadenine. The nucleic acid molecule of the present invention is
It can have a bond other than a phosphodiester bond. The nucleic acid molecule can be a peptide nucleic acid molecule. The nucleic acid molecule can be of any length and can be single-stranded or double-stranded, or partially single-stranded or partially double-stranded.

A “probe” or “probe nucleic acid molecule” is at least partially single-stranded and at least partially complementary to, or at least partially approximate to, the sequence of interest. There is a nucleic acid molecule. Probes are RNA, D
It can be NA or a combination of both RNA and DNA. Also within the scope of the invention is that the backbone sugar has a probe nucleic acid molecule that comprises a nucleic acid other than ribose or deoxyribose. The probe nucleic acid may be a peptide nucleic acid. The probe can include a label that is resistant to nuclease activity, or detectable, or can be operably linked to other moieties such as, for example, peptides.

A single-stranded nucleic acid molecule is “complementary” to another single-stranded nucleic acid molecule so that the nucleic acid molecule base pairs (hybridizes) with all or part of another nucleic acid molecule. Based on the ability of guanine (G) to base pair with cytosine (C) and adenine (A) to base pair with thymine (T) or uridine (U). Double-stranded nucleic acid molecule). For example, the nucleotide sequence 5 '
-TATAC-3 'is complementary to the nucleotide sequence 5'-GTATA-3'.

“Approximately complementary” refers to nucleic acids that selectively hybridize to one another under stringent conditions.

“Selectively hybridize” refers to a specific binding that is detectable. Polynucleotides, oligonucleotides and fragments thereof hybridize specifically with target nucleic acid strands, but under hybridization and wash conditions that minimize a significant amount of detectable binding to non-specific nucleic acids. It is. Very limiting conditions may be used to achieve the conditions of selective hybridization well known in the art. Generally, nucleic acid sequence complementarity between the polynucleotides, oligonucleotides and fragments thereof and the nucleic acid sequence of interest is at least 30%, more typically and preferably at least 40%, 50%, 60%, 70%, 80%, 90%
And can be 100%. Hybridization conditions, such as salt concentration, temperature, detergents and denaturing agents such as formamide, are varied to increase the severity of hybridization, ie, C and T which base pair with G along the nucleic acid strand, or It is possible to require an exact match for U and A for base pairing.

“Corresponding to” refers to polynucleotide sequences that share (eg, are identical) identity with all or part of a reference polynucleotide sequence. In contrast, the term "complementary to" as used herein means that the complementary sequence is base paired with all or part of a reference polynucleotide sequence. As an example, the nucleotide sequence 5'-TATAC-3 'has the reference sequence 5'-T
It corresponds to ATAC-3 'and is complementary to the reference sequence 5'-GTATA-3'.

By “sequence identity” or “identical” is meant that two polynucleotide sequences are identical (eg, on a nucleotide-by-nucleotide basis) over the range of comparison. By "partial sequence identity" or "partial identity" is meant that part of a sequence of a nucleic acid molecule is identical to at least part of the sequence of another nucleic acid molecule.

As used herein, "roughly identical" or "approximately identical" refers to a characteristic of a polynucleotide sequence, where the polynucleotide is at least 30 percent sequence identity, preferably at least 50-60. It consists of sequences having a percent sequence identity, more usually at least 60 percent sequence identity, which is compared to the reference sequence over a comparative range of at least 20 nucleotide positions, and often over a range of at least 25 to 50 nucleotides. Where, the percent sequence identity is calculated by comparing the reference sequence with a polynucleotide sequence that may contain a total of 20 percent or less of the reference sequence over the comparison range. "Approximately partial sequence identity" or "substantially identical" is used when a portion of a nucleic acid molecule is substantially identical to at least a portion of another nucleic acid molecule. "Identity" or "as used in this document
"Identical" refers to the base composition of nucleic acids, not the backbone that may consist of other components, such as one or more sugars, and one or more phosphates, or may have other substituted moieties. .

A “mutation” is a change in the genome with respect to a canonical wild-type sequence. Mutations can be deletions, insertions or transpositions of nucleic acid sequences at certain positions in the genome, and can also be single base changes at certain positions in the genome, referred to as "point mutations." Mutations can be inherited and can occur in one or more cells during the life of an individual.

“Hybridization” is the process of base pairing of single-stranded nucleic acids or single-stranded portions of nucleic acids to create double-stranded portions of double-stranded nucleic acids or nucleic acid molecules.

“Single nucleotide polymorphisms” or “SNPs” are positions of nucleic acid sequences that differ in the base composition of nucleic acids isolated from separate individuals of the same species.

“Operably linked” refers to juxtaposition, but here is a relationship that allows components as so described to function in their intended manner. For example, a control sequence, such as a promoter or enhancer or other regulatory sequence operably linked to the coding sequence, is positioned such that expression of the coding sequence is achieved under conditions comparable to the control sequences.

“Functional” in the sense of a control sequence that is functional for a nucleic acid molecule that encodes a polypeptide or protein, such as a protein involved in drug metabolism
Is in an appropriate conformation such that it is operably linked, or under appropriate conditions such as attachment of an appropriate modulator in an appropriate conformation to a control sequence,
Refers to the ability of regulatory sequences to regulate the expression of such polypeptides or proteins.

“Promoter” refers to a nucleic acid molecule, such as in DNA, to which an RNA polymerase binds to initiate transcription. Although a promoter can be regarded as a component of a gene control region, a transcription factor and a polymerase gather in the gene control region to control transcription.

“Enhancer” refers to a regulatory nucleic acid molecule, such as a DNA sequence that can influence the transcription rate of a structural gene by binding a gene regulatory protein. Included as an example of an enhancer is the GAL4 protein attached to the regulatory region of the LacZ gene, which affects the expression of beta-galactosidase. Another example is the pregnane X receptor (PXR), which regulates the transcription rate of the enzyme CYP3A4.
It binds to a DNA sequence called E or XREM. Yet another example is the Ah receptor (AhR), which binds to a specific DNA sequence called the DRE, dioxin responsive element, which sequences regulate CYPs1A1 and 1A2.
Within the regulatory domain of.

“Protein involved in drug metabolism” refers to a protein or polypeptide, and is, for example, a protein capable of regulating metabolism or metabolism of xenobiotic substances such as drugs. Included in such regulation are changes in the chemical structure of the xenobiotics via catalytic reactions and covalent or non-covalent binding, alteration of the permeability of xenobiotics in and out of cells,
Or it is the transport of xenobiotics in and out of cells.

“Drug-metabolizing enzyme” refers to an enzymatic protein, which catalyzes the modification of the covalent valence of xenobiotics such as drugs that are foreign to the host. Such change in covalent valence is optional, but is preferably an oxidation or conjugation reaction. Oxidation reactions generally result in water-soluble or increased water-soluble metabolites. For example, CYP3A4 metabolizes the drug erythromycin into a demethylated metabolite, increasing its polarity. Glucuronosyl transferase 1 (UGT1) adds glucuronide to acetaminophen to increase its polarity. CYP2C19 introduces a hydroxyl group into antiepileptic drugs to metabolize S-mephenytoin. Typically,
By increasing the polarity of xenobiotics, the modified xenobiotics are
More easily removed from the subject.

“Reporter gene” refers to a region of a nucleic acid molecule, such as DNA, that encodes a protein that is readily detected by the assay. This region can be replaced with the normal gene coding region. For example, the luciferase gene encodes a luciferase protein capable of producing a luminescent product that can be detected by a luminometer. The LacZ gene encodes a beta-galactosidase protein, which is capable of converting a particular substrate into a colored form that can be detected colorimetrically or fluorometrically in the presence of a suitable enzyme substrate. Chloramphenicol acetyltransferase (CAT) is an enzyme that metabolizes chloramphenicol and the results of this reaction can be visualized by radiometric TLC analysis.

“Intracellular receptor” refers to a polypeptide or protein that is present within a cell, where it binds molecules such as extracellular signal molecules such as ligands and initiates an intracellular response. Examples of intracellular receptors include Ah receptor or PXR.

“Hormonal receptor” refers to a steroid hormone receptor that binds to hormones that diffuse into the cell through the plasma membrane. Steroid receptors, such as thyroid hormone or vitamin D receptors, bind their ligands and then to specific DNA sequences within the genes they regulate. Examples include estrogen receptors, progesterone receptors or cortisol receptors.

“Transporter” refers to a protein within the plasma membrane that carries or otherwise diverts a molecule across the cell membrane. The transporter can be a specific transporter for a specific ligand, a general transporter for a group of ligands, an active transporter using energy such as ATP or electromotive force, or the energy of cells. Not a passive transporter. Molecules can be transported in and out of cells, depending on the transporter and the conditions in which it is placed. Examples include sodium-potassium ATPase and P-glycoprotein (MDR1), which transports drug metabolites from intracellular to extracellular.
).

“Transcription factor” refers to any polypeptide or protein that is capable of initiating or regulating transcription in cells such as, but not limited to, eukaryotic cells. Included among these are gene regulatory proteins that bind to enhancers, and common transcription factors that do not act in this specific manner. Examples of transcription factors include TFIID, common transcription factors, or specific receptors such as PXR. HNF1 is another transcription factor that regulates gene expression in a tissue-specific manner.

“Binding” in the sense of a polypeptide, such as an intracellular receptor, transporter or transcription factor, that is bound to a compound means that when the polypeptide and compound bind, the activity of the complex is dependent on the individual elements. Refers to those elements that are in contact differently than their activity.

By “operably linked” is meant the binding of one element to another, where the resulting complex can perform a function. For example, a polypeptide can bind to a compound, and the resulting complex operably binds to regulatory sequences so that expression of a gene operably linked to such regulatory sequences can be regulated. .

“Compound” means any chemical substance, test chemical substance, drug, new chemical substance (NCE
) Or other parts. For example, the compound can be a foreign substance (xenobiotic) that is not normally present in a subject, such as a mammal, including a human. The compounds can also be endogenous chemical substances that are normally present in and synthesized in biological systems such as mammals, including humans. In one embodiment, enzymatic oxidation of compounds produces generally more water soluble, easily excreted compounds. Examples include food additives, steroid hormones and drugs.

“Induce” refers to a polypeptide, such as an enzyme, such as an enzyme involved in drug metabolism in the presence of a compound, relative to the expression level of such polypeptide in the absence of the compound. Refers to increased expression. By way of example, a compound, eg, a test compound, eg, a drug, induces expression of the P450 enzyme, producing P4 produced in the presence of the compound.
Ensure that the amount of 50 enzyme is greater than the amount of P450 enzyme produced in the absence of compound.

“P450” refers to a member of the heme-containing monooxygenase superfamily involved in the catalytic oxidation of xenobiotics, such as drugs and endogenous substances such as steroid hormones. Examples include CYP2C9, CYP3A4 and C
YP1A2, but is not limited to these.

“Glucuronyl transferases” or “UGTs” refer to the polypeptides and proteins involved in glucuronidation, which is the major pathway for many lipophilic xenobiotics and endogenous substances, Promotes excretion into more water-soluble compounds. The UDP-glucuronosyltransferase family catalyzes the glucuronidation of glycosyl groups of sugar nucleotides to acceptor compounds, which are oxygen, nitrogen, sulfur, and carbon auxotrophies with formation of beta-D-glucuronide products. In the nuclear functional group. Although over 35 different UGT gene products are well known, they have been divided into two subfamilies, UGT1 and UGT2, based on sequence identity. Included in the examples are UGT1A2, UGT2B7 and UGT1A8.

“Glutathione transferase” refers to the enzyme, a soluble protein widely found in the cytosol of hepatocytes. These enzymes catalyze the conjugation of a wide variety of compounds with the endogenous tripeptide glutathione. Cytosolic glutathione S-transferases are divided into four families named alpha, mu, pie and theta, each with different but sometimes overlapping substrate specificities. There are also microsomal glutathione transferases, which are present in the endoplasmic reticulum (ER), for example. Examples include GST (mu) and GST
(Alpha), but is not limited to these.

“Sulfotransferase” refers to a polypeptide or protein such as an enzyme that catalyzes the sulfation of structurally diverse xenobiotics, including drugs and endogenous compounds. Involved in these reactions is 3'-phosphoadenosine 5'-phosphosulfate (
(PAPS) to the hydroxyl / amino group of the acceptor molecule forming sulfate and sulfamic acid, the transfer of the sulfuryl group. Sulfate conjugation is generally
Produces water-soluble metabolites and detoxifies. Sulfation is also an important element in the regulation of steroid biosynthesis, inactivation, and excretion of the endogenous hormone. Several isoforms of these enzymes exist in humans. Examples include hHST, hP-P
ST and hM-PST, but not limited thereto.

“N-Acetyltransferase” refers to a protein or polypeptide, such as an enzyme that conjugates arylamines to an acetyl group. There are distinct genes in this enzyme family. For example, NAT1 and NAT2 are N
-Encodes acetyltransferase activity. NAT1 activity is distributed in human tissues in an identical fashion, whereas NAT2 is polymorphic, allowing the detection of humans that are phenotypically slow and fast in acetylation. N-acetylation of arylamines represents a competing pathway for N-oxidation, a metabolic activation process that occurs in the liver. Heterocyclic amines are activated by acetylation by NAT2 transferase.

“P-glycoprotein” or “Pgp” refers to the MDR1 gene product. Its function is to transport drugs and steroids across cell membranes. Pgp is C
It may be a determinant of the magnitude of YP3A induction. Pgp may affect PXR ligand interactions and CYP3A-induced responses to steroids and xenobiotics.

“Enzyme” refers to a polypeptide having catalytic activity. A detectable enzyme is an enzyme that will produce a detectable product when it acts on a suitable substrate. The detectable product is preferably detected optically, eg via its emission such as fluorescence, luminescence or chemiluminescence, or by the formation of a color, eg a plastid. Recommended detectable enzymes include, but are not limited to, beta-lactamase, luciferase and beta-galactosidase.

A “detectable protein” is a polypeptide that has detectable physical properties. Recommended detectable proteins are the naturally fluorescent proteins, green fluorescent protein (GFP), SPAP renilla fluorescent protein and their derivatives.

“Extrachromosomal factor” refers to a nucleic acid molecule that, when present in a cell, does not integrate into the genome of such cell. Examples of extrachromosomal factors include plasmids and yeast artificial chromosomes (YACs).

“Intrachromosomal” in the context of a nucleic acid molecule refers to the nucleic acid molecule being within the chromosome or genome of the cell rather than being an extrachromosomal element. Nucleic acid molecules within a cell's chromosome can be "inserted" into the genome or "chromosomally internalized" by homologous recombination or other methods. When internalized to a chromosome, the nucleic acid molecule is intrachromosomal at its original locus.

To realize the phenomenon “directly” or to form a structure is to have no intermediate process or intermediate structure. For example, A and B forming AB interact directly because there is no structure between A and B. Also, C and D, which react to form E, interact directly to form E, which is
This is because there is no intermediate process between the reaction of C and D forming

“Indirectly” realizing a phenomenon or forming a structure means having an intermediate process or an intermediate structure. For example, A and B forming ABC have indirectly interacting A and B because there is a structure between A and B. Also, E and F forming G that react with H to form I are E and F
Is an indirect formation of I from, because the process of forming I involves intermediate steps.

“Cell” is any cell, eg, a prokaryotic cell or a eukaryotic cell. A cell is preferably a eukaryotic cell, preferably from a multicellular organism, but may be a unicellular organism such as yeast or a free-living eukaryotic organism. Certain cells can be obtained from organisms such as animals or humans and can be provided in primary culture, or in continuous culture as in cell lines. Although a cell may be part of a population, the population may be a population of similar cells, such as cells from the same tissue or organ, or substantially the same cells as in a clonal population of cells. And so on. The cells can be obtained from a suitable organism, eg, via conventional sampling, eg, biopsy for tissue recovery using conventional methods, or recovery of body fluids such as blood. The cells are preferably mammalian cells, preferably human cells, but need not be so. The cells are also preferably derived from a tissue that naturally exhibits relatively high levels of expression of the enzyme, although the enzyme may be a drug metabolizer such as, but not limited to, liver, intestine, lung, kidney. Get involved in. The cell may also be a transformed cell, which is a cell that has been genetically modified by a genetic engineering process such as, for example, the introduction of extrachromosomal factors or the integration of nucleic acid molecules into the chromosome of the cell.

“High throughput screening” refers to a method of screening by activity of a compound, such as a test compound such as a drug, between about 5 assays or samples per day and about 10,000 assays or sample per day. Preferably, it is performed at a rate of between about 10 assays or samples per day and about 1,000 assays or samples per day, more preferably between about 15 and about 500 assays per day.

INTRODUCTION The present invention provides improved cells and methods for identifying compounds that alter protein expression, such as chemicals and drugs. The present invention also provides other advantages.

As a non-limiting introduction to the scope of the invention, the invention includes several general and beneficial aspects including: 1) Functioning on nucleic acid molecules encoding proteins involved in drug metabolism. A cell having a first nucleic acid molecule containing a possible promoter or enhancer and a reporter gene; and a second nucleic acid encoding an intracellular receptor or transcription factor, wherein the cell is contacted with a compound having the intracellular receptor or transcription factor Or a compound is directly or indirectly activated, or is directly or indirectly regulated by a compound, an intracellular receptor or transcription factor is operably linked to a promoter or enhancer, and the reporter gene is expressed. Cells that allow; and 2) expression of genes encoding proteins involved in drug metabolism Method consisting in detecting and expression of the reporter gene; in a method of evaluating a compound for inducing properties, providing a test compound; contacting a test compound with a cell of the present invention.

These aspects of the invention, as well as others described herein, can be achieved using the methods, products and compounds of the invention. It will be appreciated that, in order to appreciate the full value of the scope of the invention, further various aspects of the invention can be combined to make a desired embodiment of the invention.

I. Cells for Evaluating Protein Expression Enhanced by Test Compound The cells contained in the present invention include a first nucleic acid molecule containing a promoter or enhancer capable of functioning with a nucleic acid molecule encoding a protein involved in drug metabolism and a reporter gene. And a second nucleic acid encoding an intracellular receptor or transcription factor, the intracellular receptor or transcription factor being operable with a promoter or enhancer when the intracellular receptor or transcription factor binds to the compound; Bind and allow the reporter gene to be expressed.

First Nucleic Acid Molecule The first nucleic acid molecule contained in the cell contains a promoter or enhancer capable of functioning in the nucleic acid molecule and a reporter gene encoding a protein involved in drug metabolism including an enzyme and a transporter. The promoter or enhancer is operably linked to the reporter gene. In this way, the reporter gene is expressed when the promoter or enhancer is activated (such as by binding the receptor / compound complex). When the reporter gene is expressed at the detection level or higher, it is considered that the promoter or enhancer is activated.

The first nucleic acid molecule is preferably double-stranded DNA, but need not be so. The first nucleic acid molecule can be extrachromosomal or intracellular in the cell.
Extrachromosomal factors include, but are not limited to, vectors, viruses, plasmids, YACs and linear nucleic acid molecules. Methods of preparing such plasmids, YACs and linear nucleic acid molecules having the properties of the first nucleic acid molecule, such as a promoter or enhancer operably linked to a reporter gene, are described in
Well known in the art. For example, nucleic acid molecules that encode promoters or enhancers that are functional to nucleic acid molecules that encode proteins involved in drug metabolism are well known in the art, and are often commercially available,
It can be prepared or cloned by conventional methods including PCR, restriction enzymes, digestion and chemical synthesis. These promoters or enhancers can be operably linked to a reporter gene using conventional methods that allow the reporter gene to be expressed when the promoter or enhancer is activated. This structure is then transformed into plasmids, viral vectors, YACs.
And into suitable vectors such as, but not limited to, linear nucleic acid molecules. These vectors can then be used to transform cells or cell populations. Such transformations are well known in the art and include electroporation, viral transmission, microprojectile bombardment, or passive uptake of nucleic acid molecules by cells.

The first nucleic acid molecule is a CMV promoter, MMTV promoter or SV4
Cells containing a nucleic acid molecule and a functional nucleic acid molecule can be selected when they contain a gene encoding a selectable marker operably linked to the promoter, such as a constitutive promoter such as the 0 promoter. A preferred selectable marker is antibiotic resistance, such that cells without such a first nucleic acid molecule are susceptible to such antibiotic, whereas cells with a functional first nucleic acid molecule are specific. Resistant to antibiotics. In this way, cells carrying the first nucleic acid molecule expressing the selectable marker can be selected and enriched. Selectable selectable markers include fluorescent proteins such as, for example, green fluorescent protein (GFP) or its derivatives, or enzymes that catalyze the formation or conversion of fluorescent substrates or products such as beta-lactamase. Under these conditions, a fluorescence activated cell sorter (FACS) can be used to isolate cells with the desired fluorescence properties.

The first nucleic acid molecule can be extrachromosomal or can integrate into the genome of the cell. When the first nucleic acid is integrated into the cell's genome, the first nucleic acid is stably integrated so that the cell is more reliable and reproducible than the transiently transfected cells. Specific vectors, such as viral vectors,
In particular, retroviral vectors can integrate into the genome. Also, homologous recombination can be used to facilitate the insertion of nucleic acid molecules into the genome of cells, as described by Treco et al., Published Feb. 13, 2001. US Pat. No. 6,187,305 to Treco e, issued May 16, 2000.
t al. , US Pat. No. 6,063,630 to US Pat. Alternatively, the transformed nucleic acid molecule can spontaneously integrate into the host genome. Integration of the first nucleic acid molecule into the cell's genome can be monitored by screening the cell for loss of a selectable marker or reporter gene, which is transiently transfected cell lines do not integrate into the cell's genome. This is because it tends to eliminate the nucleic acid molecules. Therefore, the selectable marker or reporter gene will likely tend to be lost over time through repeated passages of the cell line.

In one embodiment of the invention, the reporter gene is internal to the cell's chromosome. In this case, the reporter gene preferably encodes an enzyme, which can be readily determined by the detectable enzyme substrate or its product and the like. In this case, a nucleic acid molecule containing a promoter or enhancer functional to the desired reporter gene is genetically engineered into the vector such that integration of the vector is directed to the reporter gene or a genomic locus in its vicinity. . Incorporation of nucleic acid structures such as promoters or enhancers can be directed using homologous recombination methods known in the art, eg, Treco et al., Published Feb. 13, 2001. No. 6,187,305 to Treco et al., Issued May 16, 2000. , US Pat. No. 6,063,630. Also, naturally occurring or undirected recombinant methods known in the art can be used. Not all such homologous recombination events result in a functional linkage between the promoter or enhancer and the reporter gene, and thus the cell or population of cells is screened for such a functional linkage. Should be. For example, activation of an enhancer or promoter will lead to expression of a reporter gene if the result of the event is a functional linkage. Such expression can be monitored and screened using appropriate detectable enzyme substrates and / or products.

In another aspect of the invention, the enhancer or promoter is endogenous to the chromosome of the cell. In this case, a nucleic acid molecule containing a reporter gene that can function as an enhancer or promoter is genetically engineered into the vector such that integration of the vector is directed at or near the reporter gene at a genomic locus. The integration of the nucleic acid structure containing the reporter gene can be directed using homologous recombination methods well known in the art, which were described in February 2001 1.
Treco et al. US Pat. No. 6,187,305 to
Issue and Treco et al., Published May 16, 2000. , As described in U.S. Pat. No. 6,063,630. Not all such homologous recombination events result in a functional linkage between the promoter or enhancer and the reporter gene, and thus the cell or population of cells is screened for such a functional linkage. Should be.
For example, activation of an enhancer or promoter will lead to expression of a reporter gene if the result of the event is a functional linkage. Such expression can be monitored and screened using appropriate detectable enzyme substrates and / or products.

Second Nucleic Acid Molecule The cell also contains a second nucleic acid encoding an intracellular receptor or transcription factor.
When the intracellular receptor or transcription factor binds to the compound, the intracellular receptor or transcription factor can operably bind to the promoter or enhancer, resulting in expression of the reporter gene.

The second nucleic acid molecule is preferably, but not necessarily, double-stranded DNA. The second nucleic acid molecule can be extrachromosomal or intrachromosomal in the cell. Extrachromosomal factors include, but are not limited to, vectors, viruses, plasmids, YACs and linear nucleic acid molecules. Methods for preparing such plasmids, YACs and linear nucleic acid molecules having the properties of a second nucleic acid molecule, including nucleic acid molecules encoding intracellular receptors or transcription factors, are well known in the art. For example, nucleic acid molecules encoding intracellular receptors or transcription factors are well known in the art, are often commercially available, and can be cloned by conventional methods. This construct can then be cloned into suitable vectors such as, but not limited to, plasmids, viral vectors, YACs and linear nucleic acid molecules. These vectors can then be used to transform cells or cell populations. Such transformations are well known in the art and include electroporation, viral transmission, microprojectile bombardment, or passive uptake of nucleic acid molecules by cells.

Suitably, the second nucleic acid molecule comprises a regulatory element such as a promoter or enhancer operably linked to said nucleic acid molecule encoding an intracellular receptor or transcription factor. The regulatory element is preferably a promoter or a constitutive promoter, eg SV40 promoter, MMTV promoter or CMV.
It is a promoter. As discussed for the first nucleic acid molecule, there are art-recognized methods for creating structures such as vectors having this type of arrangement.

The second nucleic acid molecule is a CMV promoter, MMTV promoter or SV4
Cells that have taken up a nucleic acid molecule can be selected when they contain a gene encoding a selectable marker operably linked to the promoter, such as a constitutive promoter such as the 0 promoter. Preferred selectable markers include antibiotic resistance, cells without such a second nucleic acid molecule being sensitive to such antibiotic, whereas cells with a functional second nucleic acid molecule are sensitive to the particular antibiotic. Resistant. In this way, cells carrying the second nucleic acid molecule expressing the selectable marker can be selected and enriched. Selectable selectable markers include fluorescent proteins such as, for example, green fluorescent protein (GFP) or its derivatives, or enzymes that catalyze the formation or conversion of fluorescent substrates or products such as beta-lactamase. Under these conditions, a fluorescence activated cell sorter (FACS) can be used to isolate cells with the desired fluorescence properties.

In embodiments of the invention where both the first nucleic acid molecule and the second nucleic acid molecule contain a selectable marker, what is preferred, but not necessarily, is that these selectable markers are different. Different selectable markers can independently monitor both the first and second nucleic acid molecules in the cell.

The second nucleic acid molecule can be extrachromosomal or integrated into the genome of the cell. When the second nucleic acid is integrated into the genome of the cell, the second nucleic acid is stably integrated so that the cell is more reliable and reproducible than the transiently transfected cells. Specific vectors, such as viral vectors,
In particular, retroviral vectors can integrate into the genome. Also, homologous recombination can be used to facilitate the insertion of nucleic acid molecules into the genome of cells, which is described in Treco et al. US Pat. No. 6,187,305 to Treco, issued May 16, 2000
et al. , US Pat. No. 6,063,630 to US Pat. Alternatively, the transformed nucleic acid molecule can spontaneously integrate into the host genome. Integration of the second nucleic acid molecule into the cell's genome can be monitored by screening the cells for loss of a selectable marker or reporter gene, which is transiently transfected cell lines do not integrate into the cell's genome. It tends to eliminate nucleic acid molecules. Thus, selectable marker or reporter genes are likely to be lost over time. Devices and methods for incorporating nucleic acid molecules into chromosomes are well known in the art (
For example, Whitney et al. , WO98 / 13353 published on April 2, 1998; WO94 / 24301 published on October 27, 1994 at the University of Edinburgh; Treco et a published on February 13, 2001.
l. No. 6,187,305 to Treco et al., Issued May 16, 2000. , U.S. Pat. No. 6,063,630).

In one preferred embodiment of the invention, the gene encoding the intracellular receptor or transcription factor is internal to the chromosome of the cell. In particular, the gene encoding the intracellular receptor or transcription factor is in its native environment within the genome of the cell, i.e. its location and the surrounding genome, including cis-acting regulators such as promoters or enhancers. Not intentionally modified by the intervention of.

In another aspect of the invention, the gene encoding an intracellular receptor or transcription factor is endogenous to the chromosome of the cell but is a gene encoding an intracellular receptor or transcription factor such as a promoter or enhancer. A functional exogenous regulatory sequence
It is integrated into the genome of the cell and is preferably linked to the endogenous gene encoding the intracellular receptor or transcription factor and the manipulation itself. Although the integration of nucleic acid structures containing genes encoding intracellular receptors or transcription factors can be directed using homologous recombination or non-naturally directed recombination methods well known in the art. , It's Treco et, published February 13, 2001
al. No. 6,187,305 to Treco et al., Issued May 16, 2000. , As described in US Pat. No. 6,063,630.

Not all such homologous recombination events result in a functional linkage between the regulatory element and the gene encoding the intracellular receptor or transcription factor, and thus the cell or population of cells is It should be screened for such functional linkages. Such expression can be monitored and screened for using methods suitable for detecting the activity of such intracellular receptors or transcription factors. Alternatively, a reporter gene, which can be included in a construct that integrates into the genome of the cell, is operably linked to regulatory sequences used to regulate expression of intracellular receptors or transcription factors. Alternatively, the reporter gene can be operably linked to a second regulatory sequence, which regulatory sequence is the same or the same as the regulatory sequence used to regulate expression of an intracellular receptor or transcription factor. It may be different. In this case, sustained expression of the reporter gene indicates that the nucleic acid structure is operably linked to the cell's genome.

Interaction of First Nucleic Acid Molecule with Second Nucleic Acid Molecule FIG. 1 to FIG. 5 schematically show various aspects of the invention. These figures provide for the general effect of the invention in some circumstances, where the first and second nucleic acid molecules are exogenous, endogenous, integrative or extrachromosomal. Figure 1
Indicates the general interaction of the first and second nucleic acid molecules. Generally, a reporter gene is expressed when a cell is contacted with a compound that induces the expression of an enzyme or transporter involved in drug metabolism. However, when a cell does not have a gene encoding an enzyme involved in drug metabolism, or when such a gene is not in a configuration permitting expression, a protein involved in drug metabolism may not be expressed.

As shown in FIG. 1A, the second nucleic acid molecule (10) in the cell (16) contains a regulatory factor (12) and expresses a gene (14) encoding an intracellular receptor or transcription factor. Adjust. The intracellular receptor or transcription factor (18) expressed at this time can interact with the test compound (11) by an appropriate interaction such as binding, combination, regulation and the like. Test compounds can enter cells by active or passive transport mechanisms. The test compound can be optionally modified by this transport process to form a modified test compound (13). As shown in FIG. 1B,
A transcription factor or receptor can specifically bind an appropriate test compound or metabolite, which is a receptor-ligand pair, when it forms a complex (17). This complex (17) is able to associate with the first nucleic acid molecule (19) and optionally with the genome (20) of the cell. When binding to the first nucleic acid molecule (19) or to the genome of the cell (20), the complex (17) can bind to a regulatory factor that is functional to the nucleic acid molecule encoding the protein involved in drug metabolism ( 22)
, Or an endogenous regulator (24) capable of binding to such a complex (17). In the latter case, the endogenous regulator can regulate the expression of the gene (26) encoding the protein involved in drug metabolism. However, binding to endogenous regulators is not a requirement of the invention, especially in this aspect of the invention. Figure 1C
By binding the complex to a modulator of the first nucleic acid molecule, as shown in
It leads to the expression of the reporter (28) encoded by the reporter gene (30). The result of binding of the complex to endogenous regulatory sequences can lead to the expression of the endogenous protein (21) involved in drug metabolism. Endogenous proteins involved in drug metabolism can modify compound (23) via various mechanisms such as hydroxylation (25). The reporter can be detected, such as by fluorescence of the reporter (27) or conversion of the substrate (29) to a detectable substance (31) (FIG. 1D).

Thus, when compound (11) or modified compound (13) is able to bind to intracellular receptors or transcription factors (18), and the regulation of nucleic acid molecules encoding proteins involved in drug metabolism. The reporter gene (30) is expressed as a detectable reporter (28) when it is capable of binding to the sequence (22).

Proteins Involved in Drug Metabolism The proteins involved in drug metabolism can be any suitable enzyme or transporter. Preferred enzymes involved in drug metabolism include P45
0s, transporter, glucuronosyl transferase, N-acetyl transferase, glutathione transferase, p-glycoprotein and sulfotransferase. Preferred transporters include, but are not limited to, p-glycoprotein (MDR1). This protein exports drug metabolites from the cell and can affect the rate of drug metabolism by the cell. P-glycoprotein expression can be altered by certain drugs (eg, Schuetz et al., M.
ol. Pharmacol. 49: 311-318 (1999); Lan et al.
al. , Mol. Pharmacol. 58: 863-869 (2000) and Wrighton et al. , Drug Metab. Rev. 32:
339-361 (2000)). Nucleic acid molecules encoding these types of proteins have been reported and can be isolated using standard methods of molecular biology (eg, Garattini, Drug Metab. Rev. 29: 853.
-886 (1997); Schuetz et al. , Mol. Pharma
col. 49: 311-318 (1996)) and Never and D.
IETER, Pharmacology 61: 124-135 (2000)).

Promoters or Enhancers Regulatory sequences, such as promoters or enhancers, which are functional to the nucleic acid molecule encoding the protein involved in drug metabolism are preferably P450s, glucuronyl transferase, glutathione transferase and sulfotransferase or p. -A promoter or enhancer for glycoproteins. The sequences of such regulatory sequences are well known in the art and can be isolated using standard methods of molecular biology (eg, Nelson et al., DNA C.
ell Biol. 12: 1-51 (1993); Windmill et a.
l. , Mutat. Res. 376: 153-160 (1997); Schue
tz et al. J. Cell Physiol. 165: 261-272
(1995); Schuetz et al. , Mol. Pharmacol.
49: 311-318 (1996); Parker et al. J. Cli
n. Endocrin. And Metabol. 80: 1027-1031 (
1995); Blockmoller et al. , Toxicol. Let
t. 103: 173-183 (1998); Vaury et al. , Can
cer Res. 55: 5520-5523 (1995); Rodrigo e.
t al. , Scand. J. Gastroenterol. 34: 303-3
07 (1999) and Munzel et al. , Drug Metab.
Dispos. 27: 569-573 (1999)).

Reporter Gene The reporter gene can be any suitable reporter gene known in the art. The reporter gene encodes a reporter such as a detectable protein or a detectable enzyme. Detectable proteins can be detected on the basis of their physical properties, eg fluorescence in the case of fluorescent proteins such as green fluorescent protein (GFP) or its derivatives. The enzyme is detected with the appropriate substrate, which changes properties when the protein acts on the substrate to form the product. The particular substrate-enzyme combination contributes to changes in the fluorescent properties of the substrate, which, for example in the case of beta-lactamase acting on CCF2 / AM, changes the properties of FRET on the CCF2 / AM molecule.
Fluorescence can be generated in a range of glucuronidase activities on MUG. Chemiluminescence can be generated by the activity of a series of luminol dioxanes.
Luminescence can be generated by luciferase activity on luciferin (eg, Alam and Cook, Anal. Biochem. 188: 45-2.
54 (1999)). The colored product can be generated by beta-galactosidase activity on the X-Gal substrate. The potential application of reporter genes to the study of reporter gene transcription has been discussed (Alam and Cook.
, Anal. Biochem. 188: 45-254 (1990)).

Intracellular Receptors or Transcription Factors In one embodiment of the present invention, intracellular receptors or transcription factors form complexes with xenobiotics such as drugs, chemicals or their metabolites, and drugs It directly or indirectly activates a gene encoding a protein involved in metabolism. This activity is shown in the figure. According to one aspect of the invention, the intracellular receptor or transcription factor is not a hormone receptor, but it is not a requirement of the invention. According to another aspect of the invention, the intracellular receptor or transcription factor is an orphan receptor, ie a receptor with no known or recognized function. Examples of such orphan receptors include, but are not limited to, PXR and CAR (eg, Lehmann et al., J. Clin. Invests).
t. 102: 1016-1023 (1998); Jones et al. , M
ol. Endocrinol. 14: 27-39 (2000); Honko
ski et al. , Biochem. J. 347: 321-337 (200
0) and Savas et al. , Mol. Pharmacol. 56: 8
51-857 (1999)). The intracellular receptor or transcription factor can be a hormone receptor such as, but not limited to, a glucocorticoid receptor.

Cells The cells of the invention can be any cells, including prokaryotic or eukaryotic cells. The cells are preferably eukaryotic cells and are derived from mammalian subjects including humans. The cell may be of any origin, such as from mesoderm, endoderm or ectoderm. The cells can be from any tissue, organ or body fluid from the subject, but are preferably from the liver, kidneys or lungs. The cells can be provided by the subject, but can be, for example, from a sample from a biopsy or dissection, and can be well known primary cells, or can be produced by methods well known in the art. For example, a wide variety of cell lines are available from the American Type Tissue Collection (ATCC Catalog
guees (2001)). The cells can be mixed cultures of various cells or cell types are provided. For example, primary cells can include various cell types, such as hepatocytes mixed with fibroblasts. Mixed cultures of different continuous culture cell lines or mixed cultures of primary cells and continuous culture cell lines can also be used. In one embodiment of the invention, the cells are capable of being transformed and are capable of expressing an exogenous protein or polypeptide.

In one aspect of the invention, cells may be provided from a particular subject. The identity of the subject need not be known, but only the particular subject is the source of cells.
Alternatively, using cells from a population of subjects, such as those of a common racial origin, or having a common disease state, disease condition, physiological genotype or phenotype or metabolic phenotype or genotype. You can These cells can be transformed into the cells of the invention and can be used in the methods of the invention. In this case, the response of these cells to xenobiotics may indicate how the subject or population of subjects responds metabolically and physiologically to the xenobiotics. In the case of cells from a subject population, cells from different subjects can be tested separately, but need not be so. The results of these types of studies can be collected and analyzed using bioinformatics techniques aiding pharmacogenomic studies and methods. Various computer programs are available to provide such an analysis, but are not limited to such as statistical software that can provide linear or non-linear statistical techniques. The choice of statistical analysis is chosen by the person skilled in the art.

The data, analyzes and / or results produced using these methods are also part of the invention. The data, analysis and / or results can be stored on any suitable information storage medium, including, but not limited to magnetic media, tape, paper and the like. The information storage medium is preferably in a machine-readable format, but need not be so. The information storage medium may also be part of a machine, such as a machine having a central processing unit. Such machines may or may not function as part of the present invention.

II. Method for evaluating test compound for inducing expression of gene encoding protein involved in drug metabolism

The methods included in the present invention provide a test compound and contact the test compound with a cell of the invention to evaluate the compound for properties that induce expression of a gene encoding a protein involved in drug metabolism. , Detecting the expression of the reporter gene. Expression of the reporter gene indicates that the test compound has altered expression of the gene encoding the protein involved in drug metabolism. This method is possible in high throughput methods, but it is not a requirement of the invention.

Various aspects of the invention are shown in the drawings. For example, FIG. 1 shows a series of diagrams of one embodiment of the present invention in which a first nucleic acid molecule and a second nucleic acid molecule are provided as extrachromosomal elements such as plasmids. As shown in FIG. 1A, the regulatory factor P2
Regulates the transcription of genes encoding intracellular receptors or transcription factors. The translation product can then interact with a test compound that binds to intracellular receptors or transcription factors. As shown in FIG. 1B, the complex of the intracellular receptor or transcription factor and the xenobiotic or test compound may then bind to a promoter or enhancer functional to the nucleic acid molecule encoding the protein involved in drug metabolism. it can. The complex can also enter the nucleus and optionally bind to an endogenous promoter or enhancer that is functional to the nucleic acid molecule encoding the protein involved in drug metabolism, which is present or active in such cells. It is when When this complex is linked to a promoter or enhancer capable of functioning with a nucleic acid molecule encoding a protein involved in drug metabolism, a reporter gene is transcribed and translated into a reporter, and optionally an endogenous gene involved in drug metabolism. The enzyme is expressed when it is present or active in such cells (Fig. 1
C). The reporter can be a protein that is detectable by physical properties such as fluorescence, or a protein that can be detected based on enzymatic conversion of a substrate into a product such as a detectable product (FIG. 1D). According to another aspect of the invention, both the first nucleic acid molecule and the second nucleic acid molecule are provided on the same extrachromosomal element, such as a single plasmid or YAC or separate plasmids.

Alternatives to the aspects of the invention described in FIG. 1 are also provided. For example, FIG. 2 shows that the first nucleic acid molecule is an extrachromosomal factor while the second nucleic acid molecule is internal to the chromosome of the cell. FIG. 3 shows that the first nucleic acid molecule is an extrachromosomal factor and the second nucleic acid molecule is an exogenous nucleic acid molecule integrated into the cell's genome. FIG. 4 shows that the first nucleic acid molecule is an exogenous nucleic acid molecule integrated into the cell's genome and the second nucleic acid molecule is internal to the cell's chromosome. FIG. 5 shows that the first nucleic acid molecule is an exogenous nucleic acid molecule integrated into the cell's genome and the second nucleic acid molecule is an exogenous nucleic acid molecule integrated into the cell's genome. This is the case.

The method of the present invention can be practiced with suitable equipment, such as tissue culture flasks or plates having a suitable surface area per flask or well.
Suitably, the method employs a suitable plate having 6,12,24,48 or 96 wells in the footprint of a standard size microtiter plate. The method can also be used for standard footprint plates per plate of 192,288,384,480,576,672,768,864,
A dense plate with 960, 1056 or more wells. These plates are commercially available through Coster and other vendors in the commercial market.

The method can be carried out by technical personnel, or partly or wholly by robots. In the latter case, robots are used to provide high throughput,
It can reduce the cost and increase the reliability of the performance of the method. Robotic systems can be made to perform these methods. For example, sample storage units well known in the art can be used to store test compounds in an indexed fashion. A retrieval robot well known in the art is used to remove the sample from the sample storage unit and later dispensed into a test container such as a well or well of a microtiter plate using a dispensing robot well known in the art. be able to. Robots can be used to dispense cells of the invention and appropriate culture samples into test vessels using a dispensing robot well known in the art, then culture under appropriate conditions to allow such cells to To grow or maintain a culture. Incubators well known in the art can be used to provide the appropriate conditions.

Cell cultures in test vessels can be robotically combined with test compounds, such as with dispensing robots well known in the art. Cells with test compounds are provided with suitable conditions such as air and temperature, for the methods of the invention, such as incubators well known in the art. The reporter gene product can be detected directly, but by the addition of a detectable protein or enzyme substrate for the enzyme. The enzyme substrate can be added to the test container using a robot, such as a dispensing unit. The cells can be lysed, if desired, with any desired or suitable reagents that can be dispensed using robotic dispensing devices and methods well known in the art. Detection devices known in the art, such as chromogen, fluorescence, luminescence or other microtiter plate readers, can be used to detect the reporter gene product.

The output information or data generated using these methods can be directed to an information storage device such as a device that includes a central processing unit. The information storage device may also include information processing capabilities such as suitable software. The software may have the ability to perform statistical comparisons or perform statistical analysis, well known in the art, including linear and non-linear approaches.

Such robotic systems and their components are generally well known in the art and are generally described or are wholly or partially available from a variety of vendors (typically , Inventor Stylli et al. 19
(See WO98 / 52047, issued Nov. 19, 1998). The various steps and processes used to carry out the methods of the present invention can be performed independently by a robot or manually.

[0118]   Example   Example 1

[0119]   A. Equipment and methods

Plasmid Construction for Transfection The full length coding region of human PXR was derived from RNA obtained from human liver samples by RT-PCR. The oligonucleotide sequences of the forward primer and the reverse primer are 5'-ATGGAGGTGAGACCC, respectively.
AAAGAA-3 '(SEQ ID NO: 1) and 5'-CTCAGCTA
It was CCTGTGATGCCGA-3 '(SEQ ID NO: 2). PC
R conditions are 94 ° C. for 4 minutes, then 94 ° C. for 45 seconds, 55 ° C. for 1 minute and 72 ° C.
Denaturation for 30 minutes for 2 minutes and finally for 7 minutes at 72 ° C.
The 1300 base pair amplification product was cloned into pCR2.1 (Invitrog
En, Carlsbad, CA), subjected to sequence analysis. The sequence obtained corresponded to that described above (Lehmann et a, over the entire coding region.
l. J. Clin. Invest. 102: 1016-1023 (1998).
). The cDNA was then digested with BamH1 and Not1 and extracted from pCR2.1 and cloned into a similar site of the pIRES (neo) vector containing the neomycin selection cassette (Clontech, Palo Alto, CA).

Forward and reverse primers were made in the 5'-flanking region of CYP3A4, which is known to contain PXRE (Quattrochi).
et al. J. Biol. Chem. 270: 28917-28923 (
1995)). The oligonucleotide sequences of the forward primer and the reverse primer are 5'-AGACTCACCTCTGTTCAGGGGAAA-, respectively.
3 '(SEQ ID NO: 3) and 5'-CACCTTGGAAGTTTG
It was C-3 '(SEQ ID NO: 4). This 480 base pair region was PCR amplified from genomic DNA isolated from human liver samples. The amplimer was cloned into pCR2.1 and sequenced. The enhancer region was then released from pCR2.1 with EcoR1, blunt-ended, and without the mammalian selectable marker and also with the luciferase reporter gene, pGL3.
-Sma1 of the promoter vector (Promega, Madison, WI)
Cloned into the site. It was confirmed by sequence analysis that the enhancer was previously published (Quattrochi et al., J. Biol. Chem.
． 270: 28917-28923 (1995)) and that an oligonucleotide has been inserted.

[0122] G418- stably transfected with the selected HepG2 cells were resistant colonies are harvested with a saturated cell density of about 50%, 10% fetal bovine serum (FBS) 6 wells per well 5 × 10 ⁵ in DMEM containing Plates were seeded. After 24 hours of regeneration, cells were transfected at the following rates: a 5: 1 ratio of CYP3A4 enhancer / pGL3 promoter and h.
PXR / pIRES (neo) (total 6 microgram DNA / well), CY
P3A4 enhancer / pGL3 promoter and pIRES (neo) (5
: 1 ratio, 6 microgram DNA per well), pGL3 promoter and pIRES (neo) (5: 1 ratio, 6 microgram DNA / well).
, And the pGL3 promoter and hPXR / pIRES (5: 1 ratio, 6 micrograms of DNA per well) using calcium phosphate coprecipitation modification (Ausubel et al., Current Protocol).
ols in Molecular Biology, Green Publi
shing Associate / Wiley Interscience, N
ew York (1990)). Control cells have pGL3 promoter and pIRES (neo) or pGL3 promoter and pIRES (ne).
The plasmid DNA containing hPXR in o) was received. After 16 hours of exposure to the precipitated DNA, the medium was removed, cells were washed twice with DMEM and fresh medium containing 10% FBS was added. After an additional 24 hours, the medium was changed to one containing 400 micrograms of G418 per milliliter. The medium was changed every two days for three weeks until small colonies became visible. One colony was selected and transferred to a Costar 24-well plate (VWR, Westchester, PA). Each of the 24 wells contained the same medium and cells were grown to saturated cell density with medium replaced every 3 days. Wells that reached saturation cell density were trypsinized and the cells were transferred to 6-well plates, where they were further transferred to T75 flasks. Flasks that reached a saturated cell density of randomly selected colonies were trypsinized and used to seed 96-well plates to test rifampicin-induced luciferase responses of individual colonies to test for the presence of recombinants. It was measured.

Luciferase Assay The luciferase assay was performed as specified by the manufacturer (Lu
cLite system, Packard Instrument, Meri
den, CT). The activity was measured using a LumniCount luminometer manufactured by Packard, and the result was expressed as a relative luminescence amount or a fold increase relative to a control (DMSO-treated cells).

Treatment of Stable Transformed Cells HepG2-induced cell lines containing recombinant DNA are grown as monolayers in culture medium, which is Dulbecco's Modified Eagle Medium (DMEM, Gibco / BR).
L, Gaithersburg, MD), 50 U / ml penicillin, 100 micrograms streptomycin per milliliter, 0.1 mmol non-essential amino acid (Gibco / BRL), 0.4 milligrams G418 per milliliter.
Gibco / BRL) 10% fetal bovine serum (FBS, Hyclone. Loga
n, UT) and maintained at 37 ° C. in an environment of 5% CO ₂ and 95% air.
Cells were seeded in T75 flasks and grown to reach saturated cell density. After 3 to 5 days, cells were removed from the flask by trypsinization and washed with 0.1% FBS.
The cells were re-cultured in a 96-well plate at a density of about 1.0 × 10 ⁴ cells per well in DMEM medium containing G418 and G418 but no indicator (phenol red). Liver cancer, control and CY containing cells after 72 hours regeneration
P3A4 enhancers, and those with hPXR + 3A4 enhancers,
Treated with inducer dissolved in 0.1% DMSO (control) or DMSO, but at various times and concentrations in fresh medium containing 0.1% FBS and G418 but without indicator. The cells containing the CYP3A4 enhancer are identified by comparison of the results with control cells transfected with the hPXR / pIRES (neo) and pGL3 promoters or the pIRES (neo) and pGL3 promoters. Screening for cells containing the pGL3 / 3A4 enhancer is by treatment with 10 micromolar rifampicin and 0.1% DMSO. Cells showing more than a 3-fold increase in luciferase activity relative to control (DMSO treated) cells were considered to have been transformed with the correct plasmid. Finally, 3 integrated into the genome of HepG2 cells
The copy number of the A4 enhancer was confirmed by Southern blot analysis.

PIRES (neo) and pGL3 / 3A4 or hPXR + pGL3 / 3A4
A time course study was performed to test cell lines that were found to be positive in. The treatment of cells is by analysis of the response measured at 6 hour intervals for 6 to 78 hours with 10 micromolar rifampicin. In addition, various dose-response curves are displayed for various CYP3A4
Inducers and non-inducers were prepared to confirm the specificity of response factors. The dose response curve consists of concentrations ranging from 1 to 1,000 micromolar for 5 different doses. The agents tested were RU486 (Biomol, Pl
Youth Meeting, PA), Mevastatin (Biomol), Rifampicin (Sigma Chemical, St. Louis, MO), Omeprazole (Astra-Zeneca, Sweden), Clotrimazole (
Sigma), Phenobarbital (Merck, West Point, PA)
), Or dexamethasone (Sigma), and as negative inducers, pregnenolone 16 (alpha) -carbonitrile (PCN, Sigma) and T
CDD (Chemsyn Science Laboratories, Len
exa, KY). Cells were exposed to each compound for 72 hours. All inducers are DMSO (Sigma Chemical, St. Louis.
, MO) and added 0.1% of this solution to control cells.

B. Results Identification of G418-resistant colonies expressing inducible luciferase activity Stable cell lines grew on the plasmid, p3A4-enhancer, ph
By transfection of PXR and control vectors into HepG2 cells and selection from G418 resistance. Resistant colonies were identified for p3A4-enhancer, p3A4-enhancer-phPXR and control vector (Table 1). Southern blot analysis of total cellular DNA from several transformations confirmed the presence of a stably integrated CYP3A4 enhancer sequence. Verification of stable integration of hPXR into cells receiving this plasmid was performed by Northern blot analysis of several colonies (
(Figure 8). PXR mRNA showed a significant overexpression when compared to RNA from HepG2 cells not transformed with phPXR, or primary cultures of human hepatocytes. Optionally selected colonies were tested for inducible luciferase activity, but by treatment with DMSO or 10 micromolar rifampicin. Table 1 shows
Number of G418-resistant colonies screened for luciferase activity,
And the number of G418-resistant colonies with basal or induced luciferase activity.

[0127]

[Table 1]

Following transfection of HepG2 cells with the p3A4-enhancer plasmid, selection of G418 resulted in the isolation of multiple G418-resistant colonies, 13 of which were tested for luciferase activity. 11 G418
-Resistant colonies can show basal levels of luciferase expression and also 6
Colonies showed inducer-mediated luciferase activity when treated with 10 micromolar rifampicin for 48 or 72 hours.

Thirty-six G418-resistant colonies containing the stably integrated p3A4-enhancer and phPXR plasmid showed high levels of luciferase expression upon rifampicin treatment (Table 1). Three control G418-resistant colonies (clones) containing the pIRES (neo) + p luciferase plasmid showed basal levels of luciferase activity. The p3A4-enhancer + phPXR and p3A4-enhancer transformants containing the highest inducible luciferase activity were selected for further study, respectively PXR / 3A4 (colony 1F.
) And 3A4 (colony 13).

Inducible luciferase activity from stably integrated CYP3A4 sequences The first experiment performed in 96-well plates was the p3A4-enhancer + ph
It consists of reaction time curves for PXR, p3A4-enhancer, and vector control cells. Exposure to 10 micromolar rifampicin ranged from zero to 72 hours (Figure 9). In colony 3A4 / 13, rifampicin-mediated induction of luciferase activity was observed 72 to 78 hours after exposure, ranging from 35 to 43 times that of DMSO-treated cells. CYP3A4
Two distinct colonies containing enhancer and hPXR, namely colonies 1F and 6H, showed luciferase activity, which was 2.8 to 3.8 times that of DMSO treated cells exposed to 10 micromolar rifampicin for 72 hours. Was (Fig. 10). In addition, various amounts of cells were added to each well to determine the preferred or optimal amount, which, for example, showed the greatest response at the lowest background (FIG. 11). This number (50 microliters) reflects the amount of cells needed to produce a readily detectable luciferase signal, a low background level, and changes the pH of the medium over a 72 hour drug exposure period. It is an amount that does not change.

Finally, it was tested whether sera had an effect on background luciferase activity using control transformants containing the phPXR + pGL3 promoter (FIG. 12). The results also show that serum did not change luciferase activity.

Induction of CYP3A4 in Human Hepatocytes Human hepatocytes were treated with various CYP3A4 inducers for 48 hours and harvested,
Then RNA and cell homogenate were isolated. FIG. 13 shows the results of Northern blot analysis on RNA from primary cultures treated with various inducers, which include dexamethasone (10 μmol), phenobarbital (1 mmol), Rifampicin (10 μmol), clotrimazole (10 μmol), RU486 (10 μmol). The results show that cells exposed to medium without dexamethasone did not express CYP3A4. CYP3A4 levels are apparent at 0.1 and 10 micromolar dexamethasone. In fact, 10 micromolar dexamethasone significantly increased CYP3A4 mRNA by an 8-fold. In addition, rifampicin
The 3A4 message was generated in a 7.8-fold increase over that observed in cells exposed to 10 ⁻⁷ M dexamethasone in 0.1% DMSO. However, phenobarbital, clotrimazole and RU486 did not contain 0.1% DMSO and 1%.
Slightly increased CYP3A4 message was 3.8-fold, 4.9-fold and 1.7-fold, respectively, from 0 ^-7 M dexamethasone treated cells.

High Throughput System Including Stable Cell Line A 96-well plate high throughput format was used to test various inducers and non-inducers of CYP3A4. Each chemical was applied to cells at four different concentrations. Both cells containing the PXR + 3A4 enhancer and cells with the 3A4 enhancer alone (no exogenous hPXR, colony 13) were examined. 14 and 15 show the changes in luciferase activity in stably transformed cells (colony 1F), which show various well-known CYP3A4 inducers and two non-inducers, TCDD and PCN. It contains both p3A4 and phPXR treated at a single concentration. At a single concentration, omeprazole appeared to produce a maximal response when compared to other inducers, while P
CN and TCDD produced less than double the minimal luciferase activity. Colony 13 containing the 3A4 enhancer and luciferase showed a large fold increase in luciferase activity for all inducers when compared to colony 1F. Omeprazole, clotrimazole and RU486 produced maximal induction, while PCN and TCDD produced less than 1-fold increase. When three different concentrations of each inducer were tested in colony 13, 100 micromolar omeprazole produced maximal induction. Rifampicin (25 micromolar) plus 10 micromolar clotrimazole also formed between 40 and 45 fold increase (Figure 16). These results indicate that cell lines containing the CYP3A4 enhancer are effective in screening for inducers, and h during the construction of stable transformants.
The addition of PXR does not increase the induction of CYP3A4.

Example II Tested in this example is the effect of several agents such as dietary flavonoids on CYP1A1 expression using a high throughput screening system for the evaluation of human CYP induction. HepG2 cells stably integrated with the regulatory region of human CYP1A1 were treated with resveratrol, apigenin, curcumin, kaempferol, green tea extract (GTE), (−) epigallocatechin gallate (EGCG), quercetin. And by naringenin. Among these flavonoids, resveratrol produced the greatest increase (10-fold) in CYP1A1-mediated luciferase activity, while GTE, apigenin, curcumin and kaempferol produced 2- to 3-fold increase in activity. did. These flavonoids showed weak agonist activity compared to TCDD, omeprazole or benzanthracene, which had a luciferase activity increased in the range of 12 to 35 fold. The rest of the compounds, EGCG, quercetin and naringenin, showed negligible effects. Co-treatment of cells with TCDD and GTE, naringenin and apigenin was 58, 77, respectively in TCDD-mediated CYP1A1 induction.
, 74% reduction, indicating that these flavonoids show potential antagonist activity at the Ah receptor. Further results show that GTE and apigenin have Ah receptor antagonist and weak agonist activity. Further clarification is that P45 in less than 24 hours
The 96-well plate assay for 0-lead high throughput screening was effective in determining the effect of flavonoids on human CYP1A expression. Induction was easily detected in this system as the signal-to-noise ratio was low and the inter-well and replication variability was less than 10 percent. These features show that C
The reliability and feasibility of this mass screening system for the identification of YP inducers. The results obtained with the stable cell line further demonstrate that the stably integrated cell line, which is supported by primary culture of HepG2 cells and human hepatocytes and contains the enhancer factor of the P450 gene, can be used in a high throughput screening system. ing.

[0135]   A. Equipment and methods

Cell Culture and Treatment Human Liver Cancer Cell Line HepG2 (ATCC, Wistar Institute)
cell line 101L (University of Califo) derived from e)
rnia San Diego) was stably transfected with the human CYP1A1 promoter linked to a luciferase reporter gene, and a 5 ′ flanking sequence (Postlind et al., Toxicol.
Appl. Pharmacol. 118: 255-262 (1993)).
Thus, the 101L cell line is established by the stable transfection of the human hepatoma cell line HepG2 with a plasmid containing the human CYP1A1 promoter (−3275 to +89) linked to the firefly luciferase reporter gene. The CYP1A1 promoter region contains three DREs and the cell line is presumed to contain two copies of the integrated plasmid.

The 101L cell line is grown as a monolayer in medium, which is Dulbecco's Modified Eagle Medium (DMEM, Gibco / BRL), 50 U / ml penicillin, 1
00 microgram / ml streptomycin, 0.1 mmol essential amino acid (Gibco / BRL), 0.4 milligram / ml G418 (Gibco / B
RL), 10% fetal bovine serum (FBS, Hyclone, Logan UT) and maintained at 37 ° C. in an environment of 5% CO ₂ and 95% air. The cells were initially
Seed in flasks containing medium without G418. After overnight culture, the culture was changed to G418-containing medium for antibiotic selection. After 3 to 5 days, cells were removed from the flasks by trypsinization and plated at 3.5 x 105 cells per well at 2
4-well plates or have been re-cultured in 96-well plates at a cell density of wells per 7.5 × 10 ^4, which is intended to not include the G418 or indicator including 0.1% FBS (phenol red), In DMEM medium replaced with. The next day, medium containing 0.1% FBS and G418 was added to the culture. 24
After a period of time, cells containing stably integrated reporter structures were treated with 0.
1% DMSO (control), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, Chemsyn Science Laboratories)
, Lenexa, KY), 3-methylcholanthrene (3-MC, Sigma C
chemical Co. , St. Louis, Mo), Benzanthracene (B
A, Sigma), omeprazole (Astra-Zeneca, Sweden)
), Rifampicin (Rif, Sigma), quercetin (Sigma), green tea extract (GTE, Sigma), resveratrol (Sigma), apigenin (Sigma), curcumin (Sigma), kaempferol (Sigma),
With (-)-epigallocatechin gallate (EGCG, Sigma) or naringenin (Sigma) in fresh medium containing 0.1% FBS and G418 but without indicator. As an antagonist test, cells were co-treated with flavonoids and 2 nanomolar TCDD. All inducers were dissolved in DMSO and this reagent was added at 0.1% to control cells. Cells were treated with various doses and times (6 to 18 hours). After treatment, media containing compound was removed by aspiration and 100 microliter per well DM for direct analysis of luciferase activity.
Replaced with EM. Experiments were performed on 101 L cells cryopreserved at the first induction and passage number limited to 30. Later passages showed a response similar to the first passage.

Luciferase Assay Luciferase assay was performed as specified by the manufacturer (LucLit).
e system, Packard Instrument, Meriden,
CT). The activity was measured using a LumiCount luminometer manufactured by Packard, and the results were expressed as a relative luminescence amount or a control (fold increase with respect to DMSO-treated cells).

HepG2 Culture and Treatment HepG2 cells were purchased from American Type Culture Collection (ATC).
Obtained from C). Cells were grown in DMEM (Gibco / BRL). Cells were cultured and grown to saturated cell density 24 hours prior to treatment with either one of bioflavonoids, TCDD, or beta-naphthoflavone (Sigma). All inducers were dissolved in DMSO and this solution was added to control cells at 0.1%.

Primary culture and treatment of human hepatocytes The 6-well plate containing human hepatocytes was procured by the River Tissue Procurement and Distribution System (LTPADS,
University of Minnesota, Minneapolis,
MN). Upon arrival, the medium was replaced with human hepatocyte maintenance medium (HHMM, Clon
etics, San Diego, CA) (Runge et al., Bio).
chem. Biophys. Res. Commun. 273: 333-341 (
2000)) and maintained at 37 ° C. in an environment of 95% air and 5% CO ₂ . Next day, cells were incubated for 24 hours in 0.1% DMSO (control), 50
Micromolar benzanthracene, 2 nanomolar TCDD, 20 micromolar kaempferol, 20 micromolar resveratrol, or 20 micromolar naringenin, 10 micromolar apigenin, 0.1 milligram / ml.
GTE, or co-treated with TCDD and flavonoids. All inducers were dissolved in DMSO and added to the medium at a final concentration of 0.1% of this reagent. After treatment, medium was removed and cells were harvested for RNA isolation.

RNA Isolation and Northern Blot Analysis Total RNA from hepatocytes or HepG2 was isolated from Trizol® reagent (
Gibco BRL Products, Gaithersburg, Md.) And quantified by measuring absorbance at 260 nm; purity 260
The / 280 nm ratio was measured and evaluated. Northern blot analysis was performed in 1
Electrophoresis of total RNA (10 micrograms) on% agarose-2.2M formaldehyde gel followed by nylon membrane (MSI, Westboro, MA).
By blotting (Shih et al., Hum. Exper. T.
oxicol. 18: 95-105 (1999)). RNA, UV Cross
The membrane was cross-linked using a sinker (Stratagene, La Jolla, CA), the membrane encoding human CYP1A1 by the random primer method c
Hybridized with a DNA probe. A cDNA probe for human CYP1A1 has been previously described (Shih et al., Hum. Exper. To.
xicol. 18: 95-105 (1999)). A cDNA probe (Ambion, Austin TX) for the human 18S RNA probe was used to average the amount of RNA loaded in each lane. Blot hybridizations were performed as previously described (Quattrochi et al., DN.
A 4: 395-400 (1985)). Northern blot is for molecular analysis / Ma
c version 1.1.1. Image analysis software (BioRad laboratories
by means of a densitometry using a model GS-670 imaging densitometer with a Torris, Hercules, CA) or ScanM
Autoradiographs were quantified by scanning autoradiograms with an kerII (Microtek) and digitized with Un-Scan-It software (Silk Scientific, Orem Utah). The irradiation time used was within the linear range of the film, Kodak XAR-5.

Data Analysis Data were statistically analyzed using the Student's t-test. Statistical significance is p <
It was defined at the 0.05 level. Data are expressed as mean +/- standard deviation (SD).

[0143]   B. result

101 L cells are cultured at 3.5 × 10 ⁵ or 7.5 × 1 per well.
At a density of 0 ⁴ cells in 24 or 96 well plates, respectively. A
Luciferase activity was measured after exposure to the h receptor ligand benzanthracene. When comparing the results obtained from the 96-well plate assay with those from 24 plates, only a slight difference in luciferase activity was detected (Figure 17).
. These findings alleviated the concern that less cells per well would generate inappropriate signals. There was also concern that there might be large variations between replicated wells. However, the 96-well format showed up to 10% well-to-well variation in the lowest background (FIG. 17). 9
The following experiments were performed using a 6-well format.

The determination of the maximum time of inducer exposure is by setting the time course of inducer-mediated luciferase activity. The enhanced activity was found to be benzanthracene (100 μmol) and omeprazole (100 μmol).
Or within 6 hours of administration of 3-MC (10 μmol) (FIG. 18). Maximum induction by benzanthracene (35x) and 3-MC (14x) was 12
While occurring in time, omeprazole-mediated induction was maximal at 18 hours (12-fold), after which luciferase activity decreased. Induced response is reduced by He
It is likely due to metabolism of the inducer by pG2 CYP1A1. As expected, the induction by rifampicin (100 micromolar) was negligible, because the antibiotic was a CYP1A inducer (Kostrubsky e).
t al. , Drug. Metab. Dispos. 27: 887-894 (1
999)) is not known. These results indicate that this mass screening procedure is effective for monitoring easily detectable leads within a relatively short period of time, eg less than 24 hours. In addition, the concentration-dependent effects of various known CYP1A1 inducers were measured with this system. A dose response curve in the 0.5 to 2.5 nanomolar range was found for TCDD (FIG. 19A).
, And for benzanthracene and omeprazole were made from 1 to 200 micromolar (FIG. 19B). For benzanthracene and omeprazole, maximal induction (35-fold and 12-fold, respectively) occurred at 100 micromolar. The doubling induction by TCDD did not peak at the 2 nanomolar dose, and this dose showed a 22-fold increase in luciferase activity.

The use of high-throughput methods for mechanistic studies was also investigated. In order to clarify the mechanism that may be involved in flavonoid suppression of chemical carcinogenesis, we tested
The effect of some dietary flavonoids on CYP1A1 induction. The result of these studies may be whether flavonoids exhibit Ah receptor agonist and / or antagonist activity. Initial studies have examined the ability of various naturally occurring flavonoids to induce CYP1A1-promoter-mediated reporter gene activity in the 101L cell line. Dose response curves for GTE, EGCG, quercetin, curcumin, kaempferol, naringenin, apigenin and resveratrol were measured. Among these flavonoids, resveratrol (10 micromolar) produced the maximal induction (10-fold) of CYP1A1. The second most influential flavonoid inducer is apigenin,
Quercetin and curcumin (3x). A three-fold increase in luciferase activity was observed with 5 micromolar apigenin treatment, while higher doses of quercetin and curcumin (20 micromolar) provided similar levels of induction. Doses of apigenin greater than 5 micromolar produced a decrease in CYP1A1 induction, likely due to cytotoxic consequences. GTE (0.1 mg / ml) (FIG. 20, insert) and kaempferol were also at a concentration in the range of 1 to 20 micromolar, with a slight induction (2) of CYP1A1-promoter-mediated induction of luciferase activity. To 2.5-fold induction) (FIG. 20)
.

To verify a similar inducible response of the endogenous CYP1A1 gene, HepG2 cells were also treated with the same flavonoids. Enhanced CYP1A1 mRNA expression was
It was also observed in cells treated with TE (10% of TCDD induction) (Table 2).
Increased CYPA1 mRNA expression also occurred with these flavonoids, but the induction was much lower than that of beta-naphthoflavones (50% of TCDD response).
). Collectively, GTE, resveratrol and apigenin appear to be weak agonists for the Ah receptor.

[0148]

[Table 2]

The ability of flavonoids to exhibit Ah receptor antagonist activity was also examined using a high throughput screening system. Co-treatment of 101L cells with TCDD and flavonoids in a 96-well plate assay resulted in a decrease in TCDD-mediated induction of reporter gene activity by some of the flavonoids and some of these food agonists showed antagonist activity. (Fig. 21). When 101L cells were co-treated with GTE and TCDD, a 58% reduction in luciferase activity was observed compared to cells treated with TCDD alone. Furthermore, the flavonoids naringenin and apigenin produced 77% and 74% reductions in TCDD-mediated induction, respectively. The results of these studies indicate that these food flavonoids have TCD of CYP1A1 promoter activity.
It is capable of antagonizing D-mediated induction, with naringenin having greater potency (FIG. 21). Apigenin or GTE alone showed a 2.5 to 3-fold induction in reporter gene activity mediated by CYP1A1 (FIG. 20).
Based on the results, it is clear that these flavonoids show agonist and antagonist activity at the Ah receptor. Other flavonoids either did not undergo a pronounced change in the TCDD-mediated induction of luciferase activity or stimulated its effect. In fact, co-treatment with TCDD and curcumin produces 1.5-fold more stimulation than the effect of TCDD alone, and this mechanism may play a role in the induction of P450 by curcumin in addition to those involved in AhR. It shows that you cannot. HepG2 cells in TCDD
When co-treated with and individual flavonoids, results similar to those obtained with the reporter gene assay were observed. Resveratrol produced CYP1A
1 A slight decrease in the TCDD-induced response of mRNA (14% decrease), whereas apigenin and naringenin gave rise to TCDD-mediated C
There is a significant reduction in YP1A1 mRNA accumulation (57% to 70% reduction) (Table 2). These results corroborate those obtained with the 101L cell line and suggest that apigenin and naringenin have AhR antagonist activity.

To demonstrate whether similar effects occur in primary cultures of human hepatocytes, Northern analysis was performed on mRNA isolated from the cells, which showed that they were distinct from TCDD, flavonoids, or TCDD. It is processed by a combination of flavonoids. The results showed similar findings to those obtained with high throughput or high volume screening systems. Resveratrol enhanced CYP1A1 mRNA to 5% to 12% levels of TCDD induction in hepatocytes from two separate liver samples (Subject A and Subject C), while GTE was used in single cultures. In (Subject A), CYP1A1 was induced at a level of 34% of TCDD induction
mRNA was enhanced (Table 3). By comparison, 100 micromoles of benzanthracene up to 50% of what was observed in TCDD in all subjects.
Induced P1A1 mRNA. In hepatocytes from one subject, resveratrol, apigenin and kaempferol, as well as benzanthracene, caused accumulation of CYP1A1 mRNA (subject C, Table 3). Resveratrol increased the expression in 12%, apigenin in 3% and kaempferol in 10% of those observed in benzanthracene. The other two subjects (
Hepatocytes from subject B and subject D, Table 3) were CYP1A
CYP produced by TCDD and benzanthracene but did not show 1 induction
It showed 1A1 mRNA accumulation (50% of TCDD levels). Human hepatocytes were also co-treated with TCDD and individual flavonoids. Resveratrol produced a 49% reduction in the enhanced levels of TCDD-generated CYP1A1 mRNA. Apigenin and naringenin produced a 78% and 80% reduction in TCDD-mediated increase in CYP1A1 mRNA, respectively (Table 3). These results were similar to those obtained from co-treatment of the 101L cell line with TCDD and apigenin or naringenin (FIG. 21).

[0151]

[Table 3]

C. Discussion This example utilizes reporter gene assays and stable cell lines, namely 101L cells (Postlind et al., Toxicol. Ap.
l. Pharmacol. 118: 255-262 (1993)) and screen for potential CYP1A inducers. A stable cell line containing a P450 enhancer and a reporter gene is advantageous for screening applications in that
This reduces the need for sequential transfections and eliminates the variability associated with transient transfections. Stable integrated cells also significantly increase sensitivity, allowing for easily assessed induction. With consistent results, stable cells can replace other systems that are time consuming and labor intensive. Therefore, the use of stable cell lines with P450 enhancers can facilitate the screening of potential inducers. In fact, 10
The 1L reporter gene system is currently one use, CYP1A1
Is currently used by the industry in a 6-well plate format to screen environmental samples for the presence of compounds induced by L.
et al. , Environ. Toxicol. Pharmacol. 8: 1
19-126 (2000)).

To develop a high-throughput system in stable cell lines, pre-characterized 101L cells were first cultured in either 24-well or 96-well plates with standard footprints and treated with benzanthracene. (FIG. 17). The results generated from these experiments show that the 96-well plate format is as effective as the 24-well plate format. In addition, in the high throughput (96 well) format, the background was minimal and the well-to-well variation was less than 10%. Various CYP
In the presence of the 1A inducer, maximal induction (12 to 35-fold) occurred within 24 hours of exposure time and was similar to that obtained in 6-well plates (Postli).
nd et al. , Toxicol. Appl. Pharmacol. 118
: 255-262 (1993); Quattrochi and Tukey,
Mol. Pharmacol 43: 504-508 (1993)). Zicc
ardi et al. (Toxicol. Sci. 54: 183-193 (2
000)) reports a 96-well format for screening serum samples for Ah receptor ligands.

To test the high throughput format of the present invention, additional CYP1A
Inducers were tested. A dose curve was generated for benzanthracene. The maximal induction of 101L cells previously reported in 6-well plates occurred at a dose of 50 micromolar benzanthracene (Jones et al., En.
viron. Toxicol. Pharmacol. 8: 119-126 (20
00)). In the studies described herein, the same doses produced in the 96-well plate format produced the maximum activity of CYP1A1-mediated luciferase (
33 times) (Fig. 19B). Other well known CYP1A inducers, including 3-methylcholanthrene, TCDD and omeprazole, also generate induction of luciferase in a 96-well format, while rifampicin, CYP3A.
The 4 inducer was ineffective (FIG. 17), confirming the specificity of this system to respond only to the CYP1A inducer. TCDD and / or benzanthracene also
CYP1A1 mRNA was induced in HepG2 cells (Table 2), and in all human hepatocyte samples tested. Although not tested here, omeprazole has shown in previous studies to induce CYP1A's in human hepatocytes (Dias et al., Gastroenterology.
y 99: 737-747 (1990) and Shih et al. , Hum
． Expert. Toxicol. 18: 95-105 (1999)). Collectively, when one inducer produced more than a 12-fold increase in luciferase activity in a high throughput system (HTS), it was likely that CYP1 was produced by the same agent.
Induction of A1 will occur in human hepatocytes.

To determine whether this HTS could be used to identify new CYP1A1 inducing agents, the ability of various dietary flavonoids to induce CYP1A1 was examined. Of the flavonoids examined, only resveratrol produced a significant increase (10-fold) in CYP1A1-mediated luciferase activity. However, 20
Cells treated with sub-micromolar concentrations of resveratrol have negligible effect on luciferase activity, and this agent has
Previous report that it does not induce CYP1A1 mRNA in epG2 cells (
Ciolino et al. , Cancer Res. 58: 5707-57
12 (1998) and Casper, Mol. Pharmacol. 56: 7
84-790 (1999)). When the induction observed in the reporter gene assay was compared to CYP1A1 mRNA accumulation in primary hepatocytes and HepG2 cells, resveratrol, on the other hand, produced CY from HepG2 cells.
It produced an increase in P1A1 mRNA and hepatocytes from two individuals (Table 2, Table 3, particularly Subject A and Subject C). These results indicate that an agent that produces a 10-fold increase in luciferase activity found in HTS also produces CYP1A1 induction in hepatocytes. HTS system, ie G
A flavonoid that produces more than a 2.5-fold induction in TE and apigenin was identified as CYP1A1 in primary hepatocytes isolated from one of the three individuals tested here.
It also produced a slight increase in mRNA accumulation. Similarly, kaempferol, which produces a two-fold increase in luciferase activity, CYP1A1 in hepatocytes from a single individual.
Caused mRNA accumulation. In contrast, quercetin and curcumin
It did not induce CYP1A1 mRNA induction in isolated hepatocytes (data not shown), but had a slight increase in luciferase activity (2.5 to 3-fold).
Was born. Thus, such inequalities in the results between HTS and human hepatocytes among various agents suggest that reporter assays show relatively low levels of induction by certain agents. The increase in primary hepatocytes CYP1A1 (eg 2-3 fold) may or may not occur.

Based on the results obtained here with HTS, a less than 2-fold induction of luciferase activity is indicated that increased expression of CYP1A1 is unlikely in primary hepatocytes. The importance of the hepatocyte findings corroborating that of HTS lies in its ability to extrapolate human hepatocyte data to in vivo conditions (Ito et al., Ann.
u. Rev. Pharmacol. Toxicol. 38: 461-499 (1
998) and Kedderis, Chem. Biol. Interact. 1
07: 109-121 (1997)). For example, omeprazole is CYP1A '
isolated from human hepatocytes (Shih et al., Hum.Exper.T).
oxicol. 18: 95-105 (1999) and Diaz et al.
, Gastroenterology 99: 737-747 (1990)) and in vivo (Rost et al., Clin. Pharmacol. Ther.
． 52: 170-180 (1992)). In general, xenobiotics pharmacokinetics were well predicted from studies with isolated hepatocytes (
Kedderis, Chem. Biol. Interact. 107: 109-
121 (1997)). In this example, there was a perfect match between the results generated in the stably transfected cells and human hepatocytes (primary hepatocytes and HepG2 cells) and the cell lines stably transfected with the CYP enhancer. Suggest that in vivo results may be predictable.

The HTS format for assessing CYP1A1 induction is CY by the Ah receptor.
It is useful for identifying agents that can increase the expression of P1A1. Additionally, this system can be used to determine the mechanisms involved in CYP induction. What this example shows is that certain flavonoids were identified as exhibiting weak agonist and / or antagonist activity at the Ah receptor. CY
Regarding the reliability of this HTS for P inducer identification, the signal-to-noise ratio is low and the well-to-well and replication variability is less than 10%, allowing for induction readily detected in this system. The results produced by this HTS also reflect the inducer response obtained in isolated human hepatocytes or HepG2 cells.

All publications, including patent documents and scientific articles referred to in this application, including cited references, are for all purposes as if each individual publication was individually incorporated by reference. Is included by reference in its entirety.

All headings are for the convenience of the reader and should not limit the meaning of the text that follows the heading without identification.

[0160]

[Brief description of drawings]

FIG. 1 is a diagram showing that the first nucleic acid molecule and the second nucleic acid molecule are provided as an extrachromosomal factor such as a plasmid.

FIG. 2 is a diagram showing that the first nucleic acid molecule is an extrachromosomal factor, whereas the second nucleic acid molecule is endogenous to the chromosome of the cell.

FIG. 3 is a diagram showing that the first nucleic acid molecule is an extrachromosomal factor and the second nucleic acid molecule is an exogenous nucleic acid molecule integrated into the cell genome.

FIG. 4 is a diagram showing that the first nucleic acid molecule is an exogenous nucleic acid molecule integrated into the cell genome and the second nucleic acid molecule is internal to the cell chromosome.

FIG. 5 is a diagram showing that a first nucleic acid molecule is an exogenous nucleic acid molecule integrated into a cell genome, and a second nucleic acid molecule is an exogenous nucleic acid molecule integrated into a cell genome. .

FIG. 6 shows the structure of the HepG2 cell line containing a stably integrated CYP3A4 PXRE / luciferase reporter construct.

FIG. 7 shows the response of the HepG2 cell line shown in FIG.

FIG. 8 is a diagram showing Northern blot analysis of RNA.

FIG. 9 is a graph showing the effect of rifampicin and DMSO treatment on cells stably transformed with the pGL3-promoter / 3A4 enhancer.

FIG. 10 is a graph showing the effect of rifampicin treatment on cells containing phPXR in addition to the pGL3 / 3A4 enhancer.

FIG. 11. PhPXR plus pGL3 / 3A4 enhancer or varying amounts of cells containing only pGL3 / 3A4 enhancer were added to 96 well plates and treated with 10 micromolar rifampicin or DMSO for 48 hours. It is the figure which showed.

FIG. 12 is a graph showing the effect of serum, DMSO and rifampicin on luciferase activity in HepG2 cells stably transformed with pGL3 vector and pIRES vector with hPXR.

FIG. 13. Various CYP3s for CYP3A4 expression in human hepatocytes.
It is a figure showing the influence of an A4 inducer.

FIG. 14 is a graph showing the effect of various CYP3A4 inducers and non-inducers on HepG2 cells stably transformed with hPXR in pIRES vector and 3A4 enhancer in luciferase vector (colony 1F).

FIG. 15: Different CYPs for HepG2 cells stably transformed with the 3A4 enhancer in the luciferase vector (colony 13).
It is a graph which showed the influence of 3A4 inducer and a non-inducer.

FIG. 16: CYP3A4 in luciferase vector (colony 13)
FIG. 6 is a graph showing the effect of various doses of various CYP inducers on HepG2 cells stably transformed with the enhancer.

FIG. 17 is a graph showing the effect of culturing stable cell lines in 24- and 96-well plates.

FIG. 18 is a graph showing response time curves of various CYP1A1 inducers.

FIG. 19 is a graph showing response dose curves for various known CYP1A1 inducers.

FIG. 20 is a graph showing dose response curves for various flavonoids.

FIG. 21 is a graph showing the effect of co-treatment of TCDD and each flavonoid.

[Sequence list]

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Claims (20)

[Claims] 1. A first nucleic acid molecule comprising a promoter or enhancer capable of functioning in a nucleic acid molecule encoding a protein involved in drug metabolism in a cell and a reporter gene, wherein said promoter or enhancer is capable of functioning in said reporter gene. A second nucleic acid encoding an intracellular receptor or transcription factor, provided that said intracellular receptor or transcription factor binds to, is combined with, or is activated by a compound having said intracellular receptor or transcription factor, An intracellular receptor or a transcription factor, which has a promoter or an enhancer that functionally binds, combines, or is activated with the expression of the reporter gene, wherein the cell is involved in drug metabolism. With a compound that induces protein expression When you touch, cells, characterized in that said reporter gene is expressed. 2. The enzyme involved in drug metabolism is selected from the group consisting of P450s, glucuronosyl transferase, N-acetyl transferase, p-glycoprotein, glutathione tranferase and sulfotransferase. The cell according to claim 1, which comprises: 3. The cell according to claim 1, wherein the reporter gene encodes an enzyme or a detectable protein. 4. The cell according to claim 1, wherein the first nucleic acid molecule is present in an extrachromosomal factor. 5. The cell according to claim 1, characterized in that the first nucleic acid molecule is located within the chromosome of the cell. 6. The cell according to claim 1, wherein the reporter gene is inserted into the chromosome of the cell. 7. The cell according to claim 1, wherein the enhancer or promoter is endogenous to the chromosome of the cell. 8. The cell according to claim 1, wherein the reporter gene is internal to the chromosome of the cell. 9. The intracellular receptor or transcription factor forms a complex with a drug, a chemical substance or a metabolite thereof, and directly or indirectly determines the transcription activity of a gene encoding a protein involved in drug metabolism. The cell according to claim 1, which is caused by: 10. The cell according to claim 1, wherein the intracellular receptor or transcription factor is an orphan receptor or a hormone receptor. 11. The cell according to claim 1, wherein the second nucleic acid molecule is present in an extrachromosomal element. 12. The cell according to claim 1, wherein the second nucleic acid molecule is located within the chromosome of the cell. 13. The cell according to claim 1, wherein the second nucleic acid molecule is internal to the chromosome of the cell. 14. The cell according to claim 1, wherein the cell is a mammalian cell.
The cell according to. 15. The cell according to claim 1, wherein the cell is a transformed cell. 16. The cell according to claim 1, wherein the cell is a human cell. 17. The cell according to claim 1, characterized in that the cell is a cell line. 18. The cell according to claim 1, wherein the cell is derived from a tissue selected from the group consisting of liver, lung or kidney. 19. A method for evaluating a compound for the property of inducing the expression of a gene encoding a protein involved in drug metabolism, comprising the following: providing a test compound, wherein the test compound is the cell according to claim 1. Contacting and detecting expression of said reporter gene, wherein expression of said reporter gene indicates that said compound has altered expression of a gene encoding a protein involved in drug metabolism. A method characterized by. 20. The method is a high-throughput method,
The method according to claim 19.